lipid biochemistry - the journal of biological chemistry

HISTORICAL PERSPECTIVES

Lipid Biochemistry

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PROLOGUE

H1 JBC Historical Perspectives: Lipid Biochemistry. Nicole Kresge,Robert D. Simoni, and Robert L. Hill

CLASSICS

H3 The Biosynthetic Pathway for Cholesterol: Konrad Bloch

H6 The ATP Requirement for Fatty Acid Oxidation: the Early Workof Albert L. Lehninger

H8 The Kennedy Pathway for Phospholipid Synthesis: the Work ofEugene Kennedy

H11 Fatty Acid Synthesis and Glutamine Synthetase: the Work ofEarl Stadtman

H14 A Role for Phosphoinositides in Signaling: the Work of Mabel R.Hokin and Lowell E. Hokin

H16 The Selective Placement of Acyl Chains: the Work of WilliamE. M. Lands

H18 Lipid Storage Disorders and the Biosynthesis of InositolPhosphatide: the Work of Roscoe Brady

H20 The Prostaglandins, Sune Bergstrom and Bengt Samuelsson

H23 Biotin-dependent Enzymes: the Work of Feodor Lynen

H25 Acetyl-CoA Carboxylase and Other Biotin-dependent Enzymes:the Work of M. Daniel Lane

H28 The Role of the Acyl Carrier Protein in Fatty Acid Synthesis: theWork of P. Roy Vagelos

H30 How Aspirin Interferes with Cyclooxygenase Activity: the Workof William L. Smith

H33 30 Years of Cholesterol Metabolism: the Work of MichaelBrown and Joseph Goldstein

H37 Salih Wakil’s Elucidation of the Animal Fatty Acid SynthetaseComplex Architecture

H40 N-Myristoyltransferase Substrate Selection and Catalysis: theWork of Jeffrey I. Gordon

REFLECTIONS

H42 Hitler’s Gift and the Era of Biosynthesis. Eugene P. Kennedy

H55 The Biotin Connection: Severo Ochoa, Harland Wood, andFeodor Lynen. M. Daniel Lane

The Journal of Biological ChemistryTABLE OF CONTENTS

HISTORICAL PERSPECTIVES ON LIPID BIOCHEMISTRY

2010

JOURNAL OF BIOLOGICAL CHEMISTRY i

JBC Historical Perspectives: Lipid Biochemistry*

Nicole Kresge, Robert D. Simoni, and Robert L. Hill

Lipids are often broadly, and poorly, defined as biomoleculesthat are insoluble in water but soluble in organic solvents. Theyare structurally quite diverse, covering pigments, vitamins, fattyacids, cholesterol, phospholipids, sphingolipids, andmany oth-ers. The Journal of Biological (JBC) Classic articles selected forthis collection fall into two general categories: lipid biosynthesisand lipid signaling. Full accounts of the research and attributioncan be found in each Classic.

Fatty Acid Synthesis

Early work on fatty acid synthesis was done by Horace A.Barker and Earl R. Stadtman, who, in 1949, published a JBCpaper examining the synthesis of short chain fatty acids by aspecies of bacteria called Clostridium kluyveri. The pair tookadvantage of the newly available 14C isotope and used it to labelacetate and demonstrate that fatty acid synthesis is accom-plished by the multiple condensation of 2-carbon molecules.Stadtman later developed an in vitro extract to study the enzy-mology of fatty acid synthesis.The detailed enzymology of fatty acid synthesis came, in part,

from the work of P. Roy Vagelos and Salih Wakil. Using Esche-richia coli extracts in which all of the enzymes of fatty acidsynthesiswere soluble, Vageloswas able to define the individualsteps in long chain fatty acid synthesis and found that acyl car-rier protein (ACP) was the small protein to which growing acylchains were attached during the elongation cycle. He publishedhis findings in 1965 as a series of JBC papers, two of which arereproduced as JBC Classics. Wakil, who contributed to thestudies using theE. coli soluble system, alsowent on to examinefatty acid synthesis in animals, where the enzyme activitiesare part of a multifunctional enzyme complex. This work waspublished as a series of JBC papers in 1983.Adding to the studies done by Vagelos andWakil, M. Daniel

Lane, initially working with Feodor Lynen, described the firststep in fatty acid synthesis, which involves the conversion ofacetyl-CoA tomalonyl-CoA by the enzyme acetyl-CoA carbox-ylase. He also explained the role of the vitamin biotin in carbox-ylation reactions.

Phospholipid Synthesis

One of the pathways for phospholipid synthesis was deter-mined, inpart, byEugeneP.Kennedyandhis colleagues.However,Kennedy began his research career working not on lipid biosyn-thesis but on fatty acid oxidation with Albert Lehninger. In themid-1940s, theypublishedresults in the JBCthat showedthatATPwas required to “activate” fatty acids for degradation. Later,Kennedy turned his attention to synthesis reactions andmade theimportantdiscovery thatCTPis required forphospholipid synthe-

sis. He and his graduate student Samuel Weiss eventually deter-mined that intermediary formationofCDP-cholinewas occurringin the reaction.Using 14C to label the cytidine coenzymes, the pairproved that CDP-choline and cytidine diphosphate ethanolaminewere activated forms of phosphorylcholine and phosphoryleth-anolamine and were precursors of lecithin and phosphatidyleth-anolamine.This schemeof phospholipid synthesis becameknownas the “Kennedy Pathway.”In 1958, William E. M. Lands, who is best known for his

discovery of the phospholipid retailoring or “Lands” pathway,published results in the JBC that suggested “the diglyceride unitof the phospholipids is metabolically different in some respectfrom that of the triglycerides.” This initial finding led Lands topublish a series of papers in the JBC describing the selectiveplacement of acyl chains by phospholipid acyltransferases.

Cholesterol Synthesis and Regulation

The synthesis of cholesterol has a long and impressive historyin the JBC. Nobel laureate Konrad Bloch and his colleaguesmade important contributions to the understanding of the syn-thetic pathway for cholesterol, which he published in the JBC inthe mid-1940s. Eventually, through the combined efforts ofBloch, JohnCornforth, andGeorge Popjak, the origin of each ofthe 27 individual carbon atoms of cholesterol (from the methylor carboxyl group of acetate) was established. Bloch also aidedin the identification of several important landmarks in theseries of more than 30 reactions in the biosynthesis of choles-terol, including the intricate and fascinating cyclization of squa-lene to lanosterol.The regulation of cholesterol synthesis also has a history that

is reflected in the JBC.A1974paper byNobel laureates and longtime collaborators Michael S. Brown and Joseph L. Goldsteinestablished the role of LDL as a major regulator of cholesterolsynthesis. This pioneering work also revealed the existence of acell surface receptor for LDL, shed light on the process of recep-tor-mediated endocytosis, and explained the receptor defect inthe human genetic disease, familial hypercholesterolemia.

Lipid Signaling

Phosphoinostides—The role of lipid molecules in signalinghas also enjoyed a lot of coverage in the JBC. Lowell andMableHokin’s two papers, published in 1953 and 1958, demonstratedthat the acetylcholine-stimulated secretion of amylase frompancreas slices caused the incorporation of 32P into phospho-inositides. This so-called “PI Effect” laid the groundwork forthousands of studies on the role of phosphoinositides insignaling.Roscoe O. Brady studied the synthesis of inositol phospha-

tides, and in 1958, he published a JBC paper showing that anenzymesystemcatalyzed the incorporationof inositol into inositolphosphatide in the presence of Mg2� and CDP or cytidine

* To cite articles in this collection, use the citation information that appears inthe upper right-hand corner of the first page of the article.

© 2010 by The American Society for Biochemistry and Molecular Biology, Inc. Printed in the U.S.A.

JOURNAL OF BIOLOGICAL CHEMISTRY

PROLOGUE This paper is available online at www.jbc.org

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5�-phosphate. From these results, Brady proposed a mechanismfor the synthesis of inositol phosphatide in which CDP istransphosphorylated to form CDP-D-�,�-diglyceride, which thenreacts with the hydroxyl group of inositol to form inositolphosphatide.Prostaglandins—The prostaglandins are another class of

important lipid signaling molecules. Nobel laureates Sune Berg-strom andBengt Samuelsonwere pioneers in establishing the bio-synthesis and structures of several of these molecules. They pub-lished some of these studies in the JBC in themid-1960s.One of the more fascinating roles that prostaglandins play is

in mediating inflammation. In the early 1970s, John Vaneshowed that aspirin and other anti-inflammatory drugs inhibitprostaglandin synthesis. JBCAssociate EditorWilliam L. Smithwent on to show that aspirin blocks cyclooxygenase (COX1) byacylating a serine residue.

Lipid Modification of Proteins

Many proteins, particularly membrane proteins, are mod-ified by post-translational covalent lipid attachment. Jeffrey

Gordon, who has two Classic papers, has made significantcontributions to our knowledge about protein myristoyla-tion, the post-translational process by which a myristoylgroup is covalently attached via an amide bond to the �-amino group of an N-terminal glycine residue of a nascentpolypeptide.

Reflections

This collection also contains two JBC Reflections. Thefirst, titled “Hitler’s Gift and the Era of Biosynthesis,” byEugene P. Kennedy, discusses how the war brought on a flowof students to America, which caused a surge in biochemis-try research and biosynthetic studies there. In the secondarticle, M. Daniel Lane talks about his research and the cir-cumstances that brought him into contact with three greatbiochemists: Severo Ochoa, Harland Wood, and FeodorLynen.Wehope you enjoy this collection thatwe have assembled for

you.

PROLOGUE: Lipid Biochemistry

JOURNAL OF BIOLOGICAL CHEMISTRYH2

The Biosynthetic Pathway for Cholesterol: Konrad BlochOn the Utilization of Acetic Acid for Cholesterol Formation(Bloch, K., and Rittenberg, D. (1942) J. Biol. Chem. 145, 625–636)

The Utilization of Acetic Acid for the Synthesis of Fatty Acids(Rittenberg, D., and Bloch, K. (1945) J. Biol. Chem. 160, 417–424)

The Biological Conversion of Cholesterol to Pregnanediol(Bloch, K. (1945) J. Biol. Chem. 157, 661–666)

Konrad Emil Bloch (1912–2000) was born in Neisse, eastern Germany (now Nysa in Poland).Growing up he was more interested in engineering and natural sciences than chemistry, butan organic chemistry course taught by future Nobel laureate Hans Fischer at the TechnischeHochschule in Munich provided a turning point. Bloch said of Fischer’s class, “As he presentedit, the subject matter was fascinating, the organization superb, and the delivery monotonous”(1). Despite Fischer’s monotone delivery, Bloch was influenced enough to become a chemistrystudent in his laboratory.1

In early 1934, Bloch was told by Nazi authorities, in line with new racial laws, that he couldno longer study at the Technische Hochschule. Fischer managed to arrange for Bloch to workat the Schweizerisches Hohenforschung’s Institut in Davos. There, he studied the lipids ofhuman tubercle bacilli and was able to show that a previous report of the presence ofcholesterol in this organism was erroneous. This was Bloch’s first encounter with cholesterol,a subject in which he would eventually play a great role.

In 1936, with the help of R. J. Anderson at Yale University, Bloch immigrated to the UnitedStates and started working with Hans Clarke at the College of Physicians and Surgeons (P &S), Columbia University. He received his Ph.D. a year and one-half later, after completing arelatively straightforward piece of research on amino acid chemistry, and was then invited byRudolf Schoenheimer to join his group.

Also in Schoenheimer’s laboratory at this time was a scientist named David Rittenberg whohad done his graduate work on deuterium with Harold Urey. Shortly after receiving hisdegree, Rittenberg approached Schoenheimer with the prospect of using deuterium as abiological tracer. This resulted in the publication of several seminal papers authored byRittenberg and Schoenheimer on the use of deuterium to study metabolism, which was thesubject of a previous Journal of Biological Chemistry (JBC) Classic (2).

Schoenheimer was eager for Bloch to apply the isotope tracer method to study the biosyn-thesis of cholesterol and had him start by investigating whether the hydroxyl oxygen incholesterol came from water or oxygen. Unfortunately, Bloch was unable to solve this firstproblem because no method existed at that time for the mass spectrometric analysis of stablybound oxygen in complex organic compounds. Eventually, in 1956, Bloch’s student T. T. Tchenwould show that molecular oxygen is the source of the hydroxyl oxygen (3).

Schoenheimer died in 1941, and his laboratory’s research projects were divided up among itsmembers. Bloch inherited lipids, and Rittenberg acquired protein synthesis. Although he wasstill working on the cholesterol oxygen problem at that time, Bloch’s attention was quicklydiverted with the publication of a paper by R. Sonderhoff and H. Thomas, which reported that“The nonsaponifiable fraction of yeast grown in a medium supplemented with deuterated

1 All biographical information on Konrad Bloch was taken from Refs. 1, 7, 8, and 9.

THE JOURNAL OF BIOLOGICAL CHEMISTRY Vol. 280, No. 10, Issue of March 11, p. e7, 2005© 2005 by The American Society for Biochemistry and Molecular Biology, Inc. Printed in U.S.A.

ClassicsA PAPER IN A SERIES REPRINTED TO CELEBRATE THE CENTENARY OF THE JBC IN 2005

JBC Centennial1905–2005

100 Years of Biochemistry and Molecular Biology

This paper is available on line at http://www.jbc.org

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acetate had a deuterium content so high that a direct conversion of acetic acid to sterols hasto be postulated” (4). Schoenheimer and Rittenberg had also done experiments with D2O thatindicated that animal cholesterol is synthesized from small molecules (5). Combining theirareas of expertise, Rittenberg and Bloch did the next obvious experiment: they fed labeledacetates to rats and mice. As reported in the first JBC Classic reprinted here, they found thata substantial amount of deuterium was incorporated into cholesterol. However, their resultsdid not tell them how many of the 27 sterol carbon atoms were supplied by acetic acid. Thedefinitive answer came 10 years later when Bloch used an acetateless mutant of Neurosporacrassa to show that the mutant’s sterol derived all its carbon atoms from exogenous acetate (6).

Over time, Rittenberg added 15N, 13C, and 18O to the isotopes he used to study biologicalprocesses. This led to a second labeling experiment with Bloch, this time showing that aceticacid is used in the synthesis of fatty acids. This work is described in the second JBC Classic.Rittenberg and Bloch fed sodium acetate labeled with 13C and deuterium to mice and rats andfound that the animals’ lipids and cholesterol contained both labeled carbon and hydrogen.From this they concluded that both carbon atoms in acetic acid were used for the synthesis offatty acids and cholesterol.

Eventually Rittenberg became director of the isotope laboratory at P & S in 1941 andremained there until he retired. His research with isotope tracers encompassed a wide varietyof subjects, including the study of hippuric acid metabolism, the dynamics of red blood cellsurvival in patients with blood abnormalities, the development of a method to assay aminoacids in protein hydrolysates, and investigations into the synthesis of porphyrin, which will bethe subject of a future JBC Classic. Rittenberg’s many contributions to the isotope tracertechnique were recognized when he was awarded the Eli Lilly Award in Biological Chemistryfrom the American Chemical Society and also with his election to the National Academy ofSciences in 1953.2

In addition to using isotopes to study the biosynthesis of cholesterol, Bloch also used thetracers to examine the precursor role of cholesterol in bile acids and steroid hormones. In thefinal JBC Classic reprinted here, Bloch demonstrates that cholesterol is converted into pro-gesterone. However, Bloch encountered several logistical problems when starting this exper-iment. First, labeled cholesterol was unavailable commercially so he had to spend much of histime introducing deuterium into cholesterol by platinum-catalyzed exchange in heavy water-acetic acid mixtures. Second, the only practical source for isolating the progesterone metabo-lite pregnanediol in sufficient quantity was from human pregnancy urine, and his request tothe P & S department of obstetrics and gynecology for permission to administer labeledcholesterol to one of its patients was denied. Bloch eventually managed to obtain his preg-nanediol due to a “willingness to cooperate at home” (1) and proved that progesterone wasindeed synthesized from cholesterol.

In 1946 Bloch moved to the Department of Biochemistry at the University of Chicago andthen to Harvard University in 1954. He continued to study fatty acids and cholesterol as wellas the enzymatic synthesis of the tripeptide glutathione. Eventually, through the combined

2 All biographical information on David Rittenberg was taken from Ref. 10.

Konrad Bloch. Photo courtesy of the National Library of Medicine.

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efforts of Bloch, John Cornforth, and George Popjak, the origin of each of the 27 individualcarbon atoms of cholesterol (from the methyl or carboxyl group of acetate) was established.Bloch also aided in the identification of several important landmarks in the series of more than30 reactions in the biosynthesis of cholesterol, including the cyclization of squalene to lanos-terol. His work on fatty acids and cholesterol was eventually rewarded when he shared the1964 Nobel Prize in Physiology or Medicine with Feodor Lynen “for their discoveries concern-ing the mechanism and regulation of the cholesterol and fatty acid metabolism.”

The elucidation of the pathway from acetic acid to cholesterol was not only a tremendousachievement for biochemistry but also of great importance to medicine. Knowledge of thebiosynthetic pathway for cholesterol eventually aided in the discovery of statins, drugs thatinterfere with cholesterol synthesis, which are now widely used to treat high cholesterol.


REFERENCES1. Bloch, K. (1987) Summing up. Annu. Rev. Biochem. 56, 1–192. JBC Classics: Schoenheimer, R., and Rittenberg, D. (1935) J. Biol. Chem. 111, 163–168; Rittenberg, D., and

Schoenheimer, R. (1937) J. Biol. Chem. 121, 235–253 (http://www.jbc.org/cgi/content/full/277/43/e31)3. Tchen, T. T., and Bloch, K. (1956) On the mechanism of cyclization of squalene. J. Am. Chem. Soc. 78, 1516–15174. Sonderhoff, R., and Thomas, H. (1937) Ann. Chem. 530, 195–2135. Rittenberg, D., and Schoenheimer, R. (1937) Deuterium as an indicator in the study of intermediary metabolism.

XI. Further studies on the biological uptake of deuterium into organic substances, with special reference to fatand cholesterol formation. J. Biol. Chem. 121, 235–253

6. Ottke, R. C., Tatum, E. L., Zabin, I., and Bloch, K. (1951) Isotopic acetate and isovalerate in the synthesis ofergosterol by Neurospora. J. Biol. Chem. 189 429–433

7. Kennedy, E. P. (2001) Hitler’s gift and the era of biosynthesis. J. Biol. Chem. 276, 42619–426318. Goldfine, H., and Vance, D. E. (2001) Obituary: Konrad E. Bloch (1912–2000) Nature 409, 7799. Kennedy, E. P. (2003) Biographical memoirs: Konrad Bloch. Proc. Am. Philos. Soc. 147, 65–72

10. Shemin, D., and Bentley, R. (2001) Biographical Memoir of David Rittenberg, Vol. 80, pp. 256–275, NationalAcademy of Sciences, Washington, D. C.

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The ATP Requirement for Fatty Acid Oxidation: the EarlyWork of Albert L. LehningerThe Relationship of the Adenosine Polyphosphates to Fatty Acid Oxidation inHomogenized Liver Preparations(Lehninger, A. L. (1945) J. Biol. Chem. 157, 363–382)

Albert Lester Lehninger (1917–1986) was born in Bridgeport, Connecticut. In 1935 heenrolled at Wesleyan University as an English major. Although his interests soon changedto chemistry, Lehninger would later make use of his writing talents to author three classictextbooks: Biochemistry, The Mitochondrion, and Bioenergetics. Inspired by the work ofOtto Warburg and Hans Krebs, Lehninger went on to graduate school at the University ofWisconsin and received his Ph.D. in 1942. His graduate research with Edgar J. Witzemannwas on the metabolism of acetoacetate and the oxidation of fatty acids by disrupted liverpreparations.

The Journal of Biological Chemistry (JBC) Classic reprinted here concerns Lehninger’swork on fatty acid oxidation. At the time, much of what was known about glycolysis and thecitric acid cycle had been elucidated from minced tissue and tissue extracts. However, similarstudies on fatty acid oxidation had been hampered by the fact that ruptured liver cells losttheir ability to oxidize fatty acids. Luis F. Leloir and Juan M. Munoz had some success withliver homogenates at low temperatures in the presence of oxygen, inorganic phosphate,fumarate, cytochrome c, adenylic acid, and magnesium ions (1), but these experiments werenot always reproducible. When the reaction was carried out successfully, Leloir and Munoznoted that there was a decrease in ATP phosphorus and phosphopyruvic acid phosphate andan increase in inorganic phosphate, indicating the reaction was somehow coupled withphosphorylation.

In examining these early experiments, Lehninger realized that high concentrations of ATPor ADP might be required to activate or facilitate oxidation by the liver extracts. He came tothis conclusion for a number of reasons including the fact that ATP is rapidly dephosphoryl-ated when cells are disrupted and that the fumarate needed for the reaction might provide asubstrate for oxidations capable of phosphorylating adenylic acid to ATP. In the Classic,Lehninger proves his hypothesis by adding ATP to a homogenized liver preparation anddemonstrating that it consistently and reproducibly carried out fatty acid oxidation. Subse-quent work would show that fatty acids are activated by the formation of a thioester linkagebetween the carboxyl group of the fatty acid and the sulfhydryl group of coenzyme A. Thisreaction is driven by ATP.

With the start of the war, Lehninger abandoned his fatty acid studies and joined thewartime research effort of the Plasma Protein Fractionation Program led by Edwin JosephCohn, who was the author of a previous JBC Classic (2). During this time, Lehningerdiscovered several papers on oxidative phosphorylation, and from then on the mechanisms ofenergy capture and transduction in cells became the central focus of his research.

In 1945 Lehninger accepted a faculty position at the University of Chicago. During his 6years in Chicago, Lehninger and two of his students would make two significant discoveriesthat would contribute greatly to the study of metabolism. First, Lehninger and Eugene P.Kennedy would discover that virtually all of the cell’s oxidative activity occurred in themitochondria. Second, Lehninger and Morris E. Friedkin would show that electron transport

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from NADH to oxygen is an immediate and direct energy source for oxidative phosphorylation.Lehninger’s work with Kennedy and the latter part of his research career will be the subjectof a future JBC Classic.1


REFERENCES1. Munoz, J. M., and Leloir, L. F. (1943) Fatty acid oxidation by liver enzymes. J. Biol. Chem. 147, 355–3622. JBC Classics: Cohn, E. J., Hendry, J. L., and Prentiss, A. M. (1925) J. Biol. Chem. 63, 721–766 (http://www.jbc.org/

cgi/content/full/277/30/e19)3. Lane, M. D., and Talalay, P. (1986) Albert Lester Lehninger 1917–1986. J. Membr. Biol. 91, 194–197

1 All biographical information on Albert Lester Lehninger was taken from Ref. 3.

Albert L. Lehninger. Photo courtesy of the National Library of Medicine.

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The Kennedy Pathway for Phospholipid Synthesis: theWork of Eugene KennedyOxidation of Fatty Acids and Tricarboxylic Acid Cycle Intermediates by Isolated RatLiver Mitochondria(Kennedy, E. P., and Lehninger, A. L. (1949) J. Biol. Chem. 179, 957–972)

The Function of Cytidine Coenzymes in the Biosynthesis of Phospholipides(Kennedy, E. P., and Weiss, S. B. (1956) J. Biol. Chem. 222, 193–214)

Eugene Patrick Kennedy was born in Chicago in 1919. He enrolled at De Paul University in1937 as a chemistry major and then went to the University of Chicago in 1941 for graduatetraining in organic chemistry. To pay his tuition, Kennedy also got a job in the chemicalresearch department of Armour and Company, one of the large meat packers in Chicago. Aspart of the war effort, his job at Armour was to assist in the large scale fractionation of bovineblood to obtain pure bovine serum albumin. It was believed that the bovine serum albuminmight be useful for treating shock in soldiers on the battlefield. However, by the end of 1942,hope had faded that bovine serum albumin would be an effective treatment, and the Red Crossstarted to collect blood from volunteers instead. Armour opened a new facility in Fort Worth,Texas for the fractionation of human blood from donors, and Kennedy was sent to Fort Worthto assist in this effort. He remained in Texas until 1945, when the war was clearly nearing itsend and large amounts of human plasma proteins had been stockpiled.

Returning to the University of Chicago, Kennedy immediately transferred from the Depart-ment of Chemistry to the Department of Biochemistry. His experience on the plasma projecthad led to a new appreciation of biochemistry. When he was ready to begin research for hisdissertation, Kennedy approached Albert Lehninger, a young faculty member whose earlierresearch was the subject of a previous Journal of Biological Chemistry (JBC) Classic (1). Atthat time, Lehninger was studying oxidative phosphorylation and fatty acid oxidation.Kennedy writes, “With staggering naivete, I suggested to him that the proper approach wouldbe to purify the various enzymes undoubtedly involved in fatty acid oxidation and crystallizethem. He agreed that this would be desirable, but went on to point out rather gently that fattyacid oxidation had not yet been demonstrated in a soluble extract from which individualenzymes might be isolated. To reach that stage, it would first be necessary to discover thenature of the energy-requiring activation or ”sparking“ of fatty acid oxidation and the specialdependence of the process on particulate structures” (2).

Despite this initial incident, Lehninger agreed to take Kennedy on as a graduate student,and he began to work on the problem of fatty acid oxidation in 1947. Lehninger had observedthat both fatty acid oxidation and oxidative phosphorylation were inhibited in a strikinglyparallel fashion when particulate enzyme preparations of homogenized rat livers were exposedto hypotonic buffers. The activity could be preserved by adding either salts or iso-osmoticamounts of sucrose to the buffers.

Kennedy’s first project in the laboratory was a detailed study of these effects (3). Thesestudies led Lehninger and Kennedy to surmise that fatty acid oxidation, oxidative phospho-rylation, and the Krebs cycle must all be taking place in one organelle, bounded by amembrane impermeable to certain solutes. Although their enzyme preparations were quitecrude, they were convinced that the organelle was the mitochondrion, even though function-ally and morphologically intact mitochondria had not yet been isolated.

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Around this time George Palade and his collaborators were developing methods for theseparation and identification of organelles. As reported in a previous JBC Classic (4), Paladeworked out a method for the isolation of purified mitochondria by differential centrifugation in0.88 M sucrose. Kennedy immediately tested mitochondria isolated by this method and ob-tained convincing evidence that oxidative phosphorylation, fatty acid oxidation, and thereactions of the Krebs cycle did indeed occur in the mitochondria. This is the subject of the firstJBC Classic reprinted here.

After finishing graduate school, Kennedy went to the University of California, Berkeley, towork with Horace A. Barker. Barker and his graduate student Earl Stadtman, both of whomwill be featured in future JBC Classics, had just discovered that soluble extracts of Clostrid-ium kluyveri cells could produce short-chain fatty acids from ethyl alcohol. Although the initialdiscovery had already been made, there was much to be learned about these extracts andKennedy aided in this effort.

In 1950, Kennedy joined Fritz Lipmann, author of a previous JBC Classic (5), at HarvardMedical School.1 He then returned to the University of Chicago in 1951, after being given ajoint appointment in the Department of Biochemistry and the newly organized Ben MayLaboratory for Cancer Research.

In Chicago, Kennedy started to study the origins of the phosphodiester bond of phos-phatidylcholine using labeled choline. He found that free choline, but not phosphocholine,was converted to lipid in a reaction dependent on ATP generated by oxidative phosphoryl-ation (6). At the same time, Kornberg and Pricer (7) reported experiments in whichphosphocholine was converted to a lipid (later identified as lecithin) in a reaction thatrequired ATP. Determined to understand why he and Kornberg had obtained contradictingresults, Kennedy, along with his graduate student Samuel Weiss, undertook a detailedexamination of the differences between the two studies. They discovered that they couldreproduce Kornberg’s results using commercially available ATP. However, large amountsof ATP were needed, suggesting that an impurity, rather than ATP, might be involved inthe reaction.

Kennedy and Weiss’ discovery of the cofactor involved in the conversion of phosphocholineto lecithin is the subject of the second JBC Classic reprinted here. After testing severalnucleoside triphosphates, they realized that cytidine triphosphate (CTP) was the activecofactor in the phosphocholine reaction. They formulated a number of schemes to accountfor the involvement of CTP in phospholipid synthesis and eventually decided that inter-mediary formation of cytidine diphosphate choline (CDP-choline) was occurring in thereaction. Although they had no evidence for its involvement, they synthesized CDP-cholineand cytidine diphosphate ethanolamine and tested their abilities to act as cofactors in lipidbiosynthesis. Using 14C to label the cytidine coenzymes, Kennedy and Weiss proved thatCDP-choline and cytidine diphosphate ethanolamine were activated forms of phosphoryl-choline and phosphorylethanolamine and were precursors of lecithin and phosphatidyleth-anolamine. They also showed that the two cytidine coenzymes were present in highquantities in liver and yeast.

In 1959, Kennedy was invited to become a Hamilton Kuhn Professor and head of theDepartment of Biological Chemistry at the Harvard Medical School. He continued his researchon phospholipid biosynthesis and was able to formulate a detailed picture of the pathways ofbiosynthesis of the principal glycerophosphatides and of triacylglycerol by 1961. Kennedy’sinterests also led him to investigate membrane biogenesis and function in bacteria, thetranslocation of membrane phospholipids, and periplasmic glucans and cell signaling inbacteria. Kennedy is currently at Harvard as the Hamilton Kuhn Professor of BiologicalChemistry and Molecular Pharmacology, Emeritus.2


REFERENCES1. JBC Classic: Lehninger, A. L. (1945) J. Biol. Chem. 157, 363–382 (http://www.jbc.org/cgi/content/full/280/14/e11)2. Kennedy, E. (1992) Sailing to Byzantium. Annu. Rev. Biochem. 61, 1–283. Lehninger, A. L, and Kennedy, E. P. (1948) The requirements of the fatty acid oxidase complex of rat liver. J. Biol.

Chem. 173, 753–771

1 Please see Ref. 8 for Kennedy’s JBC Reflection on Fritz Lipmann, Rudolf Schoenheimer, and Konrad Bloch.2 All biographical information on Eugene P. Kennedy was taken from Ref. 2.

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4. JBC Classic: Hogeboom, G. H., Schneider, W. C., and Palade, G. E. (1948) J. Biol. Chem. 172, 619–635(http://www.jbc.org/cgi/content/full/280/22/e19)

5. JBC Classic: Lipmann, F. (1945) J. Biol. Chem. 160, 173–190 (http://www.jbc.org/cgi/content/full/280/21/e18)6. Kennedy, E. P. (1953) The synthesis of lecithin in isolated mitochondria. J. Am. Chem. Soc. 75, 249–2507. Kornberg, A., and Pricer, W. E. (1952) Fed. Proc. 11, 2428. Kennedy, E. P. (2001) Hitler’s gift and the era of biosynthesis. J. Biol. Chem. 276, 42619–42631

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Fatty Acid Synthesis and Glutamine Synthetase: the Workof Earl StadtmanFatty Acid Synthesis by Enzyme Preparations of Clostridium kluyveri. I. Prepara-tion of Cell-free Extracts That Catalyze the Conversion of Ethanol and Acetate toButyrate and Caproate(Stadtman, E. R., and Barker, H. A. (1949) J. Biol. Chem. 180, 1085–1093)

Allosteric Regulation of the State of Adenylylation of Glutamine Synthetase inPermeabilized Cell Preparations of Escherichia coli. Studies of Monocyclic andBicyclic Interconvertible Enzyme Cascades, in Situ(Mura, U., Chock, P. B., and Stadtman, E. R. (1981) J. Biol. Chem. 256, 13022–13029)

Earl Reece Stadtman was born in 1919 in Carrizozo, a small town in New Mexico. When hewas 10, his family moved to San Bernardino, California, where he attended high school. Aftergraduating from high school in 1937, Stadtman enrolled in several science courses at SanBernardino Valley College, hoping to eventually set up a soil-testing laboratory. However, hesoon realized that he needed a more rigorous education and enrolled at the University ofCalifornia, Berkeley. He earned a B.S. in soil science in 1942.

After spending a year in Alaska, involved in a wartime project of mapping the Alaskan-Canadian (Al-Can) Highway, Stadtman returned to Berkeley looking for work. He paid a visitto Horace A. Barker, a Berkeley biochemist for whom he had worked as a laboratory technician(and author of a future Journal of Biological Chemistry (JBC) Classic). At that time, Barkerwas directing various war efforts in the Department of Food Technology and offered Stadtmana job as principal investigator on a project studying the “Browning of Dried Apricots,” the goalof which was to find a way to slow the deterioration of dried fruits during storage. Around thistime, Stadtman also met his future wife, Thressa Campbell, who was working as a laboratoryassistant in the food technology department.

After the war, Stadtman started graduate studies in the Department of Biochemistryworking in Barker’s laboratory. Barker had spent a year as a postdoc in Albert J. Kluyver’slaboratory at the Technical School in Delft in the Netherlands before coming to Berkeley andhad isolated a species of bacteria called Clostridium kluyveri (named after Kluyver) from theDelft canal mud. Since then, Barker had been searching for an explanation for the observationthat C. kluyveri could produce short-chain fatty acids from ethyl alcohol. He made a break-through when he obtained some 14C and used the isotope to label acetate and demonstrate thatfatty acid synthesis is accomplished by the multiple condensation of 2-carbon molecules (1). Hededuced that ethanol is first oxidized to “active” acetate (a 2-carbon compound), which iscondensed with acetate to form a 4-carbon compound that is reduced to form butyrate. Activeacetate can then be condensed with butyrate to form caproate. Barker surmised that acetylphosphate might be the active acetate formed in the above reaction.

It was at this point that Stadtman joined Barker’s laboratory and started working on fattyacid synthesis. Initially, like Barker, Stadtman used 14C to trace the metabolic pathways inwhole cell preparations of C. kluyveri. However, he abandoned this approach after a visit toIrwin C. Gunsalus’s laboratory at Cornell University. Gunsalus showed Stadtman how to drybacterial cells and grind the dried preparations to break open cell walls, producing a cell-freeextract. Applying this method to C. kluyveri, Stadtman was able to produce extracts that couldcatalyze all of the reactions involved in the conversion of ethanol and acetate to fatty acids of

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4- and 6-carbon atoms. His preparations also catalyzed the aerobic oxidation of ethanol andbutyrate. These experiments are reported in the first JBC Classic reprinted here. Thisdiscovery was especially significant because up until that time most biochemists believed thatthe capacity to make fatty acids was a unique property of specialized cellular systems orparticulate organelles.

In a series of additional papers (2–6), all published in the JBC, Stadtman and Barker usedthe enzyme extracts to study the individual reactions involved in fatty acid synthesis andconfirmed that ethanol is oxidized to acetyl phosphate, which condenses with acetate andforms butyric acid. They also discovered that C. kluyveri contained an acetyl-transferringenzyme (phosphotransacetylase) and an enzymatic system for using acetyl phosphate toactivate other fatty acids. Stadtman later showed that acetyl-CoA was the source of activeacetate in the synthesis of butyric acid from acetyl phosphate (7) while working as postdoctoralfellow with Fritz Lipmann (author of a previous JBC Classic (8)).

In 1950, Stadtman began to look for an academic position. However, because his wifeThressa also had a Ph.D., they were looking for an institution at which they could both workat the same professional level. Unfortunately, at that time, most universities had anti-nepotism rules that did not allow more than one family member to work in the samedepartment. Intended to protect universities from charges of favoritism, the rules often hadthe effect of discriminating against married women. No one seriously challenged the rulesuntil the 1960s, when the American Association of University Women began to protest theirunfairness. Fortunately, these polices were not in effect at the National Institutes of Health(NIH), and in September 1950, the Stadtmans moved to Bethesda, Maryland. Both continue todo research at the NIH today.

At the NIH, Stadtman continued his research on fatty acid metabolism. In 1952, hesuccessfully carried out the first in vitro net synthesis of acetyl-CoA using only basic materials(acetyl phosphate, CoA, and phosphotransacetylase). Stadtman and his postdoc P. Roy Vagelosalso demonstrated that long-chain fatty acid synthesis is catalyzed by an enzyme complex inwhich malonyl-CoA is the source of active acetate.

Another topic of long term research in Stadtman’s laboratory was glutamine synthetase, theenzyme that catalyzes the conversion of glutamate to glutamine. The activity of glutaminesynthetase is subject to feedback inhibition by 7 different end products of glutamine metab-olism. Stadtman discovered that this end product inhibition was cumulative (the presence ofmore end products resulted in more inhibition) and that susceptibility to feedback inhibition

Photo courtesy of the Office of NIH History, National Institutes of Health.

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only occurred when glutamine synthetase was adenylated by adenylyltransferase (ATase). Helater found that adenylation was regulated by uridylyltransferase (UTase), which, dependingon the cellular concentration of various metabolites, catalyzed the covalent attachment of auridylyl group to the regulatory protein, PII. The uridylated form of PII stimulates ATase tocatalyze glutamine synthetase deadenylation, whereas the unmodified form of PII stimulatesATase catalysis of the adenylation reaction.

In view of these results, Stadtman surmised that glutamine synthetase activity was con-trolled by a cascade system in which two systems of reversible covalent modification weretightly linked. Each system was composed of two reversible reactions, or two interconvertibleenzyme cycles, the linkage of which resulted in the formation of a bicyclic cascade system. Thiscascade system allowed enzyme activity to be shifted gradually in response to metaboliteavailability.

In the late 1970s and early 1980s, Stadtman and P. Boon Chock carried out a theoreticalanalysis of this bicyclic cascade system to understand its implications in enzyme regulation.However, it was not until 1981 that they were able to study the cascade in vivo. Theseexperiments are discussed in the second JBC Classic reprinted here. Stadtman had discoveredthat after a freeze-thaw cycle, treatment of Escherichia coli cells with a nonionic detergentrendered them permeable to small metabolites but allowed the cells to retain the proteincomponents of the cascade system. Furthermore, permeabilized cells from cultures containing10 mM glutamine retained all their cascade enzymes whereas 5 mM glutamine-grown cells hadinactivated UTase. Using these cells, they were able to study the effects of different substratesand allosteric effects on the cascade system and to confirm the previous theoretical and in vitrostudies. A more complete description of Stadtman’s work on glutamine synthetase can befound in his JBC Reflections (9).

Stadtman has received many awards and honors for his numerous research discoveriesincluding the 1979 National Medal of Science, the 1983 ASBC-Merck Award, and the 1991Robert A. Welch Award in Chemistry. Stadtman was also President of the American Societyfor Biological Chemists (now American Society for Biochemistry and Molecular Biology) from1982 to 1983 and has been a member of the National Academy of Sciences since 1969.1


REFERENCES1. Barker, H. A., Kamen, M. D., and Bornstein, B. T. (1945) The synthesis of butyric and caproic acids from ethanol

and acetic acid by Clostridium kluyveri. Proc. Natl. Acad. Sci. U. S. A. 31, 373–3812. Stadtman, E. R., and Barker, H. A. (1949) Fatty acid synthesis by enzyme preparations of Clostridium kluyveri.

II. The aerobic oxidation of ethanol and butyrate with the formation of acetyl phosphate. J. Biol. Chem. 180,1095–1115

3. Stadtman, E. R., and Barker, H. A. (1949) Fatty acid synthesis by enzyme preparations of Clostridium kluyveri.III. The activation of molecular hydrogen and the conversion of acetyl phosphate and acetate to butyrate. J. Biol.Chem. 180, 1117–1124

4. Stadtman, E. R., and Barker, H. A. (1949) Fatty acid synthesis by enzyme preparations of Clostridium kluyveri.IV. The phosphoroclastic decomposition of acetoacetate to acetyl phosphate and acetate. J. Biol. Chem. 180,1169–1186

5. Stadtman, E. R., and Barker, H. A. (1949) Fatty acid synthesis by enzyme preparations of Clostridium kluyveri.V. A consideration of postulated 4-carbon intermediates in butyrate synthesis. J. Biol. Chem. 181, 221–235

6. Stadtman, E. R., and Barker, H. A. (1950) Fatty acid synthesis by enzyme preparations of Clostridium kluyveri.VI. Reactions of acyl phosphates. J. Biol. Chem. 184, 769–794

7. Stadtman, E. R., Novelli, G. D., and Lipmann, F. (1951) Coenzyme A function in and acetyl transfer by thephosphotransacetylase system. J. Biol. Chem. 191, 365–376

8. JBC Classic: Lipmann, F. (1945) J. Biol. Chem. 160, 173–190 (http://www.jbc.org/cgi/content/full/280/21/e18)9. Stadtman, E. R. (2001) The story of glutamine synthetase regulation. J. Biol. Chem. 276, 44357–44364

10. Park, B. S. The Stadtman way: A tale of two biochemists at NIH. http://history.nih.gov/exhibits/stadtman/index.htm (An online exhibit produced by the Office of NIH History in collaboration with the National Heart,Lung, and Blood Institute)

1 All biographical information on Earl R. Stadtman was taken from Ref. 10.

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A Role for Phosphoinositides in Signaling: the Work ofMabel R. Hokin and Lowell E. HokinEnzyme Secretion and the Incorporation of P32 into Phospholipides of Pancreas Slices(Hokin, M. R., and Hokin, L. E. (1953) J. Biol. Chem. 203, 967–977)

Phosphoinositides and Protein Secretion in Pancreas Slices(Hokin, L. E., and Hokin, M. R. (1958) J. Biol. Chem. 233, 805–810)

Mabel R. Hokin (born Mabel Neaverson) and Lowell E. Hokin met in Hans Kreb’s depart-ment at the University of Sheffield and married soon after. During their time in Sheffield, theHokins started investigating what they thought was an increase in the incorporation of 32Pinto RNA caused by the acetylcholine-induced stimulation of pancreatic slices. However,before they could purify the RNA, they moved to McGill University in Montreal, Quebec, andbrought their radiolabeled samples with them. Once established in Montreal, they continuedwith their experiments but noticed that as they purified the RNA the radioactivity was lost.Investigating this phenomenon further, they found that most of the radioactivity was incor-porated into the phospholipid fraction. This was a surprising discovery as up until thenphospholipids were regarded as inert structural components of membranes.

The Hokins’ studies on 32P uptake into phospholipids during enzyme secretion in pancreasslices are published in the first Journal of Biological Chemistry (JBC) Classic reprinted here. TheHokins incubated pigeon pancreas slices with various compounds along with 32P to see the effectson phosphate incorporation into phospholipids. They found that when enzyme secretion wasstimulated by acetylcholine or carbamylcholine, both of which induce amylase secretion, theincorporation of 32P into phospholipids was on average 7.0 times greater than in control tissue.

Separating individual phospholipids for analysis was difficult at that time, but fortunately RexDawson devised a method that permitted the analysis of diacylglycerophospholipids by deacyla-tion and two-dimensional separation of the water-soluble backbone (1). The Hokins used thismethod to show that hormone stimulation of pancreatic slices mainly increased the rate of 32Pincorporation into phosphoinositide but that phosphatidylcholine, phosphatidylserine, and phos-phatidic acid also contained radiolabeled phosphate (2). This was the first demonstration ofreceptor-stimulated lipid turnover, and it later became known as the “PI effect.”

The second JBC Classic reprinted here presents the details of the Hokins’ study of phos-phoinositide metabolism in relation to protein secretion in the pancreas. They incubatedpigeon pancreas slices with either NaH2P32O4, [2-3H]inositol, or [1-14C]glycerol and extractedthe lipids from the tissue and separated them by paper chromatography. They were able toidentify seven phospholipids containing 32P as well as two radioactive monophosphoinositides.From these data they concluded, “the present work indicates that phosphoinositides areinvolved in the secretion of protein from the inside of the pancreatic acinar cell into thelumen . . . It is tempting to think that the active transport out of the cell of many other typesof molecules may involve phosphoinositides.”

In 1957 the Hokins moved to Madison, Wisconsin, where they both joined the faculty of theUniversity of Wisconsin-Madison Medical School. There they showed that other tissues exhibitsimilar responses when provoked to secrete. In 1964 the Hokins suggested that phospholipaseC-catalyzed phosphatidylinositol hydrolysis might initiate the PI effect. Later it was confirmedthat the initiating event was the phospholipase C-catalyzed hydrolysis of phosphatidylinositol

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4,5-bisphosphate and that 3-kinase-catalyzed formation of phosphatidylinositol 3,4,5-triphos-phate was a second widespread signaling reaction.

The Hokins’ initial work on stimulated phosphoinositide turnover in secretory tissuesmotivated a large number of other investigators to focus their research on the PI effect andsecond messengers. Eventually they would discover that the Hokins’ inositol phospholipidsplay important roles in transmembrane signaling and many other cell regulatory processes.1


REFERENCES1. Dawson, R. M. C. (1954) The measurement of 32P labeling of individual kephalins and lecithin in a small sample

of tissue. Biochim. Biophys. Acta 14, 374–3752. Hokin, L. E., and Hokin, M. R. (1955) Effects of acetylcholine on the turnover of phosphoryl units in individual

phospholipids of pancreas slices and brain cortex slices. Biochim. Biophys. Acta 18, 102–1103. Michell, B. (2003) Obituary: Mabel R. Hokin (1924–2003). The Biochemist. December4. Irvine, R. F. (2003) 20 years of Ins(1,4,5)P3, and 40 years before. Nat. Rev. Mol. Cell. Biol. 4, 586–590

1 All biographical information on Mabel R. Hokin and Lowell E. Hokin was taken from Refs. 3 and 4.

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The Selective Placement of Acyl Chains: the Work ofWilliam E. M. LandsMetabolism of Glycerolipids: A Comparison of Lecithin and Triglyceride Synthesis(Lands, W. E. M. (1958) J. Biol. Chem. 231, 883–888)

William E. M. Lands was born in Chillicothe, Missouri in 1930. He earned his B.S. inchemistry from the University of Michigan in 1951 and his Ph.D. in biological chemistry fromthe University of Illinois in 1954. After graduating, he spent a year as a postdoctoral fellow atthe California Institute of Technology and then joined the faculty of the University of Michiganas an instructor in biological chemistry. Lands spent the next 25 years at Michigan, eventuallybecoming professor in 1967.

In 1980, Lands left Michigan to head the Department of Biological Chemistry at theUniversity of Illinois. He spent 10 years there and then moved to Bethesda, Maryland tobecome Senior Scientific Advisor to the Director of the National Institute on Alcohol Abuse andAlcoholism (NIH). In 2002, Lands retired from his position at the NIH.

Lands spent the majority of his scientific career studying fatty acids and has made manysignificant contributions to this field. One such contribution is his discovery of the phospho-lipid retailoring or “Lands” pathway. His initial paper showing the likelihood of acyl chainturnover is reprinted here as a Journal of Biological Chemistry (JBC) Classic.

In the paper, Lands incubates various tissues with [14C]acetate and [14C]glycerol andmeasures the value R, which is the ratio of [14C]acetate to [14C]glycerol in the diglyceride unitof the triglycerides and phospholipids produced by the tissues. Lands reasoned that if diglyc-

William E. M. Lands

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eride is the sole precursor of phos-pholipids, these two compoundsshould have the same R value. Also,because a third fatty acid molecule isadded to the diglyceride unit to formtriglycerides, they should have a 3/2R value (see Fig. 1).

However, Lands’ results showedthat R is 2–4 times higher in phos-pholipids than in triglycerides, sug-gesting to him that “the diglycerideunit of the phospholipids is metabol-

ically different in some respect from that of the triglycerides.” This initial finding led to a seriesof papers published in the JBC describing the selective placement of acyl chains by phospho-lipid acyltransferases (1–4).

In addition to the above research which helped to explain the metabolic process thatregulates the mixture of acyl chains found in lipids, Lands is also credited with discovering thebeneficial effects of balancing excess �-6 fatty acids with dietary �-3 fatty acids.

In recognition of his contributions to science, Lands received the Glycerine Research Award(1969), the Canadian Society of Nutritional Science Lectureship (1991), and the American OilChemists’ Society Supelco Lipid Research Award (1997). The University of Michigan’s Depart-ment of Biological Chemistry has also endowed a lectureship in honor of Lands. He has alsoserved on the editorial boards of several journals, including those of the Journal of LipidResearch, Biochimica et Biophysica Acta, Lipids, and Prostaglandins.


REFERENCES1. Lands, W. E. M. (1960) Metabolism of glycerolipids. II. The enzymatic acylation of lysolecithin. J. Biol. Chem. 235,

2233–22372. Lands, W. E. M., and Merkl, I. (1963) Metabolism of glycerolipids. III. Reactivity of various acyl esters of coenzyme

A with ��-acylglycerol phosphorylcholine and positional specificities in lecithin synthesis. J. Biol. Chem. 238,898–904

3. Merkl, I., and Lands, W. E. M. (1963) Metabolism of glycerolipids. IV. Synthesis of phosphatidylethanolamine.J. Biol. Chem. 238, 905–906

4. Lands, W. E. M., and Hart, P. (1965) Metabolism of glycerolipids. VI. Specificities of acyl-CoA:phospholipidacyltransferases. J. Biol. Chem. 240, 1905–1911

FIGURE 1

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Lipid Storage Disorders and the Biosynthesis of InositolPhosphatide: the Work of Roscoe BradyThe Enzymatic Synthesis of Inositol Phosphatide(Agranoff, B. W., Bradley, R. M., and Brady, R. O. (1958) J. Biol. Chem. 233, 1077–1083)

Roscoe Owen Brady was born in 1923 in Philadelphia. He attended Pennsylvania StateUniversity from 1941 to 1943 and then received his medical degree from Harvard MedicalSchool in 1947. After interning at the Hospital of the University of Pennsylvania for 1 year,Brady did a postdoctoral fellowship in the Department of Physiological Chemistry at theUniversity of Pennsylvania School of Medicine (1948 to 1950) and then was a fellow in clinicalmedicine in the Department of Medicine (1950 to 1952). In 1954, following 21⁄2 years on activeduty in the U.S. Naval Medical Corps, he joined the National Institutes of Health (NIH) tobecome section chief of the National Institute of Neurological Diseases and Blindness. Heremained in this position until 1967 when he became assistant laboratory chief of neurochem-istry at the National Institute of Neurological Diseases and Blindness. Currently, Brady isChief of the Developmental and Metabolic Neurology Branch of the National Institute ofNeurological Disorders and Stroke, a position he has held since 1972.

Early in his career at the NIH, Brady started studying lipids, specifically inositol and thesynthesis of inositol phosphatide. This is the subject of the Journal of Biological Chemistry(JBC) Classic reprinted here. Although inositol had been isolated more than 100 years beforethe Classic was published, little was known about its metabolism. In his Classic, Brady usestritium-labeled inositol and a preparation from guinea pig kidney mitochondria to studyinositol metabolism. He found that the enzyme system catalyzed the incorporation of inositolinto inositol phosphatide in the presence of Mg2� and cytidine diphosphate-choline (CDP) orcytidine 5�-phosphate. From these results, Brady proposed a mechanism for the synthesis ofinositol phosphatide in which CDP is transphosphorylated to form CDP-D-�,�-diglyceride,which then reacts with the hydroxyl group of inositol to form inositol phosphatide.

These early studies stimulated Brady’s interest in lipid storage disorders, in particular,Gaucher disease. In 1967, he showed that people with Gaucher disease had low levels ofglucocerebrosidase and thus were unable to break down the lipid glucocerebroside and clear itout of their bodies. He also developed a diagnostic test for Gaucher disease, which worked bymeasuring glucocerebrosidase activity in white blood cells. A year later, Brady suggested atherapy for Gaucher disease based on replacing the enzyme. Using human placentas, his teamisolated a tiny sample of purified glucocerebrosidase and gave it to two patients. The patients’health improved, and Brady soon developed large scale purification and targeting methods forglucocerebrosidase to use in further clinical trials. Eventually, in 1991, Brady’s macrophage-targeted glucocerebrosidase enzyme replacement therapy was approved as a specific treat-ment for Gaucher disease by the Food and Drug Administration.

In addition to studying Gaucher disease, Brady has discovered the metabolic basis ofNiemann-Pick disease, Fabry disease, and the specific biochemical defect in Tay-Sachs dis-ease. He has applied this knowledge to developing diagnostic tests, carrier identificationprocedures, and the prenatal detection of such conditions. Currently his research is focused onexamining enzyme replacement therapy and gene therapy for patients with these otherhereditary metabolic disorders.

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In recognition of his scientific achievements, Brady received the Gairdner InternationalAward (1973), the Cotzias Award from the American Academy of Neurology (1980), thePassano Foundation Award (1982), the Lasker Foundation Clinical Medical Research Award(1982), and the Kovalenko Medal from the National Academy of Sciences (1991). He is amember of the National Academy of Sciences and a member of the Institute of Medicine of theNational Academy of Sciences. He has served on the editorial boards and advisory boards ofmany journals and organizations.



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The Prostaglandins, Sune Bergstromand Bengt SamuelssonProstaglandins and Related Factors. 15. The Structures of Prostaglandin E1,F1�, and F1�

(Bergstrom, S., Ryhage, R., Samuelsson, B., and Sjovall, J. (1963) J. Biol. Chem. 238,3555–3564

On the Mechanism of the Biosynthesis of Prostaglandins E1 and F1�

(Hamberg, M., and Samuelsson, B. (1967) J. Biol. Chem. 242, 5336–5343)

Sune Karl Bergstrom (1916–2004) was born in Stockholm, Sweden. Upon completing highschool he went to work at the Karolinska Institute as an assistant to Erik Jorpes where he didresearch on the biochemistry of fats and steroids. Jorpes was sufficiently impressed withBergstrom that in 1938 he sponsored a year-long research fellowship for him at the Universityof London. Then, in 1940, Bergstrom received a Swedish-American Fellowship, which allowedhim to study for 2 years at Columbia University and to conduct research at the SquibbInstitute for Medical Research in New Jersey. He returned to Sweden in 1942 and receiveddoctorates in medicine and biochemistry from the Karolinska Institute 2 years later. He wasthen appointed assistant in the biochemistry department of Karolinska’s Medical NobelInstitute.

Bergstrom’s involvement with prostaglandins started in 1945 at a meeting of the Physio-logical Society of the Karolinska Institute. There he met Ulf von Euler who had been doingresearch on prostaglandins. Von Euler asked Bergstrom if he might be interested in studyingsome of his lipid extracts of sheep vesicular glands. Using Lyman Craig’s countercurrentextraction device, which was the subject of a previous Journal of Biological Chemistry (JBC)Classic (1), Bergstrom was able to purify the crude extract about 500 times. However, his workwas interrupted for a few years when he was appointed chair of physiological chemistry at theUniversity of Lund in 1948.

When Bergstrom resumed his research on prostaglandins, he was aided by his graduatestudent Bengt Ingemar Samuelsson. Samuelsson, who was born in Halmstad, Sweden, in1934, had enrolled at the University of Lund to study medicine when he came under thementorship of Bergstrom. Using countercurrent fractionations and partition chromatography,Bergstrom was able to isolate small amounts of prostaglandin E1 and F1� by 1957 (2). A yearlater, Bergstrom was appointed professor of chemistry at Karolinska, and he moved hisresearch group with him to Stockholm. Samuelsson received his doctorate in medical sciencefrom the Karolinska Institute in 1960 and his medical degree in 1961.

At Karolinska, Bergstrom started to collaborate with Ragnar Ryhage who had built acombination gas chromatograph and mass spectrometer. Using this instrument, Bergstrom,Samuelsson, and Ryhage were able to deduce the structures of prostaglandins E1, F1�, and F1�

from mass spectrometric identification of the products formed when the prostaglandins weretreated with a weak acid or base. These structure determinations are discussed in the firstClassic reprinted here. By 1962, Bergstrom and his colleagues had isolated and determinedthe structures of six different prostaglandins.

After completing the structural work on the prostaglandins, Samuelsson spent a year as apostdoctoral fellow with E. J. Corey in the Department of Chemistry at Harvard University,where he was able to study theoretical and synthetic organic chemistry. He returned to the

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Karolinska Institute as assistant professor of medical chemistry and resumed work on theprostaglandins. The second JBC Classic deals with some of Samuelsson’s research on thebiosynthesis of prostaglandins, an area in which he contributed considerable knowledge. Inthe paper, Samuelsson follows the conversion of 8,11,14-eicosatrienoic acid to prostaglandin E1and prostaglandin F1�, using 3H and 14C labeling, focusing especially on the initial step of theprocess.

In 1967, Samuelsson joined the faculty of the Royal Veterinary College in Stockholm asProfessor of Medical Chemistry to explore the veterinary and livestock breeding applicationsof prostaglandins. However, he returned to the Karolinska Institute in 1972 to becomeProfessor and Chairman of the Department of Physiological Chemistry. He was Dean of theMedical Faculty from 1978 to 1983 after which he was appointed Rector of the KarolinskaInstitute.

Bergstrom remained at Karolinska, serving as dean of its medical school from 1963 to 1966and as Rector of the Institute from 1969 to 1977. He was chairman of the Nobel Foundation’sBoard of Directors from 1975 to 1987, and from 1977 to 1982 he served as chairman of theWorld Health Organization’s Advisory Committee on Medical Research. He retired fromteaching in 1981, choosing to devote his full time to research at Karolinska.

Independently, both Bergstrom and Samuelsson continued to investigate prostaglandinsand related compounds throughout their scientific careers. In honor of their contributions tothis field, they were awarded the 1982 Nobel Prize in Physiology or Medicine with John R.Vane “for their discoveries concerning prostaglandins and related biologically activesubstances.”

Samuelsson’s research has been recognized by numerous awards and honors in addition tothe Nobel Prize. These include the A. Jahres Award in Medicine from Oslo University (1970),the Louisa Gross Horwitz Prize from Columbia University (1975), the Albert Lasker MedicalResearch Award (1977), the Ciba-Geigy Drew Award for biomedical research (1980), theGairdner Foundation Award (1981), the Bror Holberg Medal of the Swedish Chemical Society(1982), and the Abraham White Distinguished Scientist Award (1991). Samuelsson waselected to the National Academy of Sciences in 1984.1

Bergstrom also received many awards, including the Albert Lasker Award in 1977, OsloUniversity’s Anders Jahre Prize in Medicine in 1970, and Columbia University’s Louisa GrossHorwitz Prize in 1975. He was a member of the Royal Swedish Academy of Science (and served

1 Biographical information on Bengt Samuelsson was taken from Ref. 3.

Sune Karl Bergstrom. Photo courtesy of theNational Library of Medicine.

Bengt Ingemar Samuelsson. Photo courtesy ofthe National Library of Medicine.

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as its president from 1983 to 1985), the American Philosophical Society, the National Academyof Sciences (1973), and the American Academy of Arts and Sciences.2


REFERENCES1. JBC Classics: Craig, L. C. (1943) J. Biol. Chem. 150, 33–45; Craig, L. C. (1944) J. Biol. Chem. 155, 519–534

(http://www.jbc.org/cgi/content/full/280/7/e4)2. Bergstrom, S., and Sjovall, J. (1957) The isolation of prostaglandin. Acta Chem. Scand. 11, 10863. Samuelsson, B. I. (1993) Studies of biochemical mechanisms to novel biological mediators: prostaglandin endoper-

oxides, thromboxanes and leukotrienes. In Nobel Lectures, Physiology or Medicine 1981–1990 (Frangsmyr, T.,ed) World Scientific Publishing Co., Singapore

4. Bergstrom, S. K. (1993) The prostaglandins: from the laboratory to the clinic. In Nobel Lectures, Physiology orMedicine 1981–1990 (Frangsmyr, T., ed) World Scientific Publishing Co., Singapore

2 Biographical information on Sune Bergstrom was taken from Ref. 4.

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Biotin-dependent Enzymes: the Work of Feodor LynenThe Enzymatic Synthesis of Holotranscarboxylase from Apotranscarboxylase and(�)-Biotin. I. Purification of the Apoenzyme and Synthetase; Characteristics ofthe Reaction(Lane, M. D., Young, D. L., and Lynen, F. (1964) J. Biol. Chem. 239, 2858–2864)

Feodor Felix Konrad Lynen (1911–1979) was bornin Munich, Germany. He was undecided about hiscareer during his early education and even consid-ered becoming a ski instructor. Ultimately, he en-rolled in the Department of Chemistry at the Uni-versity of Munich where he studied with Nobellaureate Heinrich Wieland and received his doctor-ate degree in 1937. Three months later, he marriedWieland’s daughter, Eva.

After graduating, Lynen remained at MunichUniversity as a postdoctoral fellow. He was ap-pointed lecturer in 1942 and assistant professor in1947. When World War II broke out, Lynen wasexempt from military service because of a kneeinjury resulting from a ski accident in 1932. How-ever, the war made it difficult to continue to doresearch in Munich, and Lynen moved his labora-tory to the small village of Schondorf on the Am-mersee. This was lucky because in 1945 MunichUniversity’s Department of Chemistry was de-stroyed. Lynen continued his work at various lab-

oratory facilities and eventually returned to the rebuilt Department of Chemistry in 1949.During the 1940s, Lynen began studying the biosynthesis of sterols and lipids. He eventu-

ally initiated a collaboration with Konrad Bloch, whose cholesterol research was featured in aprevious Journal of Biological Chemistry (JBC) Classic (1). Working together, Bloch andLynen were able to elucidate the steps in the biosynthesis of cholesterol. An especiallysignificant finding made by Lynen was that acetyl coenzyme A (previously discovered by JBCClassic author Fritz Lipmann (2)) was essential for the first step of cholesterol biosynthesis.Lynen later determined the structure of acetyl-CoA. This work on cholesterol resulted in Blochand Lynen being awarded the 1964 Nobel Prize in Physiology or Medicine.

In 1953, Lynen was made full professor at the University of Munich. A year later, he wasnamed director of the newly established Max Planck Institute for Cell Chemistry. He contin-ued to work on fats but also turned his focus to biotin-dependent enzymes. In 1962, he wasjoined by JBC Classic author M. Daniel Lane (3), who had come to Munich to work with Lynenon a sabbatical leave. Lane was studying the biotin-dependent propionyl-CoA carboxylase andhad previously determined that its biotin prosthetic group was linked to the enzyme throughan amide linkage to a lysyl �-amino group.

Before leaving for Munich, Lane developed an apoenzyme system with which to investigatethe mechanism by which biotin became attached to propionyl-CoA carboxylase. This systemmade use of Propionibacterium shermanii, which expressed huge amounts of methylmalonyl-

Feodor Lynen

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CoA:pyruvate transcarboxylase, another biotin-dependent enzyme. The organism also had anabsolute requirement for biotin in its growth medium and produced large amounts of theapotranscarboxylase when grown at very low levels of biotin.

As reported in the JBC Classic reprinted here, Lane and Lynen were able to resolve andpurify both the apotranscarboxylase and the synthetase that catalyzed biotin loading onto theapoenzyme. Dave Young, a postdoctoral fellow who had recently completed his medicaltraining at Duke University, collaborated with them on these studies. In a second paperreprinted in the Lane Classic (4), Lane and Lynen showed that the synthetase catalyzed atwo-step reaction. The first step involved the ATP-dependent formation of biotinyl-5�-AMP andpyrophosphate after which the biotinyl group was transferred from the AMP derivative to theappropriate lysyl �-amino group of the apotranscarboxylase. Lane and Lynen also showed thatthe covalently bound biotinyl prosthetic group, like free biotin, was carboxylated on the 1�-Nposition (5).

In 1972, Lynen moved to the recently founded Max Planck Institute for Biochemistry.Between 1974 and 1976, he was acting director of the Institute while continuing to oversee alab at the University of Munich. He remained at the Institute until his death in 1979.

In addition to the Nobel Prize, Lynen received many honors and awards. These include theNeuberg Medal of the American Society of European Chemists and Pharmacists (1954), theLiebig Commemorative Medal of the Gesellschaft Deutscher Chemiker (1955), the CarusMedal of the Deutsche Akademie der Naturforscher Leopoldina (1961), and the Otto WarburgMedal of the Gesellschaft fur Physiologische Chemie (1963).


REFERENCES1. JBC Classics: Bloch, K., and Rittenberg, D. (1942) J. Biol. Chem. 145, 625–636; Rittenberg, D., and Bloch, K. (1945)

J. Biol. Chem. 160, 417–424; Bloch, K. (1945) J. Biol. Chem. 157, 661–666(http://www.jbc.org/cgi/content/full/280/10/e7)

2. JBC Classics: Lipmann, F. (1945) J. Biol. Chem. 160, 173–190 (http://www.jbc.org/cgi/content/full/280/21/e18)3. JBC Classics: Lane, M. D., Rominger, K. L., Young, D. L., and Lynen, F. (1964) J. Biol. Chem. 239, 2865–2871;

Gregolin, C., Ryder, E., Warner, R. C., Kleinschimdt, A. K., Chang, H.-C., and Lane, M. D. (1968) J. Biol. Chem.243, 4236–4245; Guchhait, R. B., Polakis, S. E., Hollis, D., Fenselau, C., and Lane, M. D. (1974) J. Biol. Chem. 249,6646–6656; Polakis, S. E., Guchhait, R. B., Zwergel, E. E., Lane, M. D., and Cooper, T. G. (1974) J. Biol. Chem. 249,6657–6667 (http://www.jbc.org/cgi/content/full/281/49/e40)

4. Lane, M. D., Rominger, K. L., Young, D. L., and Lynen, F. (1964) The enzymatic synthesis of holotranscarboxylasefrom apotranscarboxylase and (�)-biotin. II. Investigation of the reaction mechanism. J. Biol. Chem. 239,2865–2871

5. Lane, M. D., and Lynen, F. (1963) The biochemical function of biotin. VI. Chemical structure of the carboxylatedactive site of propionyl carboxylase. Proc. Natl. Acad. Sci. U. S. A. 49, 379–385

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Acetyl-CoA Carboxylase and Other Biotin-dependentEnzymes: the Work of M. Daniel LaneThe Enzymatic Synthesis of Holotranscarboxylase from Apotranscarboxylase and(�)-Biotin. II. Investigation of the Reaction Mechanism(Lane, M. D., Rominger, K. L., Young, D. L., and Lynen, F. (1964) J. Biol. Chem. 239,2865–2871)

Liver Acetyl-CoA Carboxylase. II. Further Molecular Characterization(Gregolin, C., Ryder, E., Warner, R. C., Kleinschimdt, A. K., Chang, H.-C., and Lane,M. D. (1968) J. Biol. Chem. 243, 4236–4245)

Acetyl Coenzyme A Carboxylase System of Escherichia coli. Site of Carboxylation ofBiotin and Enzymatic Reactivity of 1�-N-(Ureido)-Carboxybiotin Derivatives(Guchhait, R. B., Polakis, S. E., Hollis, D., Fenselau, C., and Lane, M. D. (1974) J. Biol.Chem. 249, 6646–6656)

Acetyl Coenzyme A Carboxylase System of Escherichia coli. Studies on the Mecha-nisms of the Biotin Carboxylase- and Carboxyltransferase-catalyzed Reactions(Polakis, S. E., Guchhait, R. B., Zwergel, E. E., Lane, M. D., and Cooper, T. G. (1974)J. Biol. Chem. 249, 6657–6667)

Malcolm Daniel Lane was born in Chicago in 1930. He received both his B.S. and M.S. fromIowa State University in 1951 and 1953, respectively. Lane then went to the University ofIllinois for graduate school and was awarded his Ph.D. in 1956. He joined the faculty of theVirginia Polytechnic Institute and State University in Blacksburg, Virginia in 1956 as Asso-ciate Professor and was promoted to Professor of Biochemistry in 1963.

Upon joining the faculty at Virginia Polytechnic Institute, Lane decided to try to determinehow propionate was metabolized in the bovine liver. About this time, Journal of BiologicalChemistry (JBC) Classics author Severo Ochoa (1, 2) reported that propionyl-CoA was carbox-ylated to form methylmalonyl-CoA, which was then was converted to succinyl-CoA. Lane wasable to purify propionyl-CoA carboxylase from bovine liver mitochondria.

Then in 1959 a paper by Lynen and Knappe appeared in Angewandte Chemie (3) indicatingthat �-methylcrotonyl-CoA carboxylase, a biotin-dependent carboxylase, catalyzed the ATP-dependent carboxylation of “free” biotin in the absence of its acyl-CoA substrate. Lynenproposed that the free biotin had accessed the active site of the carboxylase and by mimickingthe biotinyl prosthetic group it had been carboxylated. Lane determined that propionyl-CoAcarboxylase was also a biotin-dependent enzyme and determined that the biotin prostheticgroup was linked to propionyl-CoA carboxylase through an amide linkage to a lysyl �-aminogroup.

In 1962 Lane decided to take a sabbatical leave in Munich with Feodor Lynen at theMax-Planck Institut Fur Zellchemie where he continued to work on the enzymatic mechanismby which biotin became attached to propionyl-CoA carboxylase. Before leaving for Munich,Lane developed an apoenzyme system with which to investigate the “biotin loading” reaction.This system made use of Propionibacterium shermanii, which expressed huge amounts ofmethylmalonyl-CoA:pyruvate transcarboxylase, another biotin-dependent enzyme. The orga-nism also had an absolute requirement for biotin in its growth medium and produced largeamounts of the apotranscarboxylase when grown at very low levels of biotin.

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In Munich, Lane was able to resolve and purify both the apotranscarboxylase and thesynthetase that catalyzed biotin loading onto the apoenzyme (4). Dave Young, a postdoctoralfellow who had recently completed his medical training at Duke University, and KarlRominger, a Ph.D. candidate under Lynen’s direction, collaborated with him on these studies.In a second paper, which is the first JBC Classic reprinted here, Lane and his colleaguesshowed that the synthetase catalyzed a two-step reaction. The first step involved the ATP-dependent formation of biotinyl-5�-AMP and pyrophosphate after which the biotinyl group wastransferred from the AMP derivative to the appropriate lysyl �-amino group of the apotrans-carboxylase. Lane and Lynen also showed that the covalently bound biotinyl prosthetic group,like free biotin, was carboxylated on the 1�-N position (5).

Shortly after he returned from Munich, Lane left Virginia Polytechnic Institute to becomeAssociate Professor of Biochemistry at the New York University School of Medicine. He waslater promoted to Professor of Biochemistry in 1969. In New York, Lane and his colleaguesisolated acetyl coenzyme A carboxylase from chicken liver (6). The biotin-containing enzymecatalyzes the carboxylation of acetyl-CoA to malonyl-CoA in a 2-step process involving acarboxybiotin intermediate. In an accompanying paper, Lane described the molecular char-acteristics of the enzyme, including its reversible inter-conversion between protomeric andpolymeric forms. The paper is reprinted here as the second JBC Classic by Lane. He deter-mined that the carboxylase has a binding site for citrate and another for acetyl-CoA and thatcitrate binding might be involved in regulating the enzyme.

Lane left New York in 1970 to become Professor of Biological Chemistry at the JohnsHopkins University School of Medicine. Right around the time Lane took up his new post atJohns Hopkins, Thomas C. Bruice and A. F. Hegarty published a paper (7) that called intoquestion Lane’s conclusion that biotin was carboxylated on the 1�-N position. They pointed outthat carboxylation could occur at the ureido-O and result in the same derivative. In the thirdJBC Classic, Lane uses the acetyl coenzyme A carboxylase system from Escherichia coli toprovide definitive evidence that the ureido-N of biotin is the site of carboxylation.

In the final JBC Classic reprinted here, Lane presents a thorough analysis of the acetylcoenzyme A carboxylase system from Escherichia coli. He defines the requirements andproperties of isotopic exchange and stoichiometric reactions representative of the two half-reactions in acetyl-CoA carboxylation and also describes studies using prosthetic group andintermediate model derivatives as substrates to elucidate the mechanisms of the partialreactions.

Lane was eventually promoted to Director and DeLamar Professor in 1978. He is currentlyDistinguished Service Professor in the Department of Biological Chemistry at Johns Hopkins.More information about Lane’s early work on biotin can be found in his JBC Reflections (8).

Lane’s honors and awards include the American Institute of Nutrition’s Mead-Johnsonaward in 1966, the American Society of Biological Chemists’ William C. Rose award in 1981,and the Johns Hopkins University School of Medicine Professor’s Award for Distinction in

M. Daniel Lane

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Teaching in 1986. He was elected to the American Academy of Arts and Sciences in 1982, theAmerican Society for Nutritional Sciences in 1996, and the National Academy of Sciences in1987. In addition to serving as president of the American Society for Biochemistry andMolecular Biology in 1990, Lane served on the Society’s Program Committee, MembershipCommittee, and Public Affairs Committee. He has served on the Editorial Boards of severaljournals, including those of the Journal of Biological Chemistry, Biochemistry et BiophysicaActa, the Archives Biochemistry and Biophysics, and Annual Reviews of Biochemistry. He alsoserved on the editorial board and was Executive Editor of Biochemical and BiophysicalResearch Communications in 1986.


REFERENCES1. JBC Classics: Stern, J. R., and Ochoa, S. (1951) J. Biol. Chem. 191, 161–172; Korkes, S., del Campillo, A.,

Gunsalus, I. C., and Ochoa, S. (1951) J. Biol. Chem. 193, 721–735 (http://www.jbc.org/cgi/content/full/280/11/e8)2. JBC Classics: Salas, M., Smith, M. A., Stanley, W. M., Jr., Wahba, A. J., and Ochoa, S. (1965) J. Biol. Chem. 240,

3988–3995 (http://www.jbc.org/cgi/content/full/281/21/e16)3. Lynen, F., Knappe, J., Lorch, E., Jutting, G., and Ringelmann, E. (1959) Die biochemische Funktion des Biotins.

Angew. Chem. 71, 481–4864. Lane, M. D., Young, D. L., and Lynen, F. (1964) The enzymatic synthesis of holotranscarboxylase from apotrans-

carboxylase and (�)-biotin. I. Purification of the apoenzyme and synthetase; characteristics of the reaction.J. Biol. Chem. 239, 2858–2864

5. Lane, M. D., and Lynen, F. (1963) The biochemical function of biotin. VI. Chemical structure of the carboxylatedactive site of propionyl carboxylase. Proc. Natl. Acad. Sci. U. S. A. 49, 379–385

6. Gregolin, C., Ryder, E., and Lane, M. D. (1968) Liver acetyl coenzyme A carboxylase. I. Isolation and catalyticproperties. J. Biol. Chem. 243, 4227–4235

7. Bruice, T. C., and Hegarty, A. F. (1970) Biotin-bound CO2 and the mechanism of enzymatic carboxylationreactions. Proc. Natl. Acad. Sci. U. S. A. 65, 805–809

8. Lane, M. D. (2004) The biotin connection: Severo Ochoa, Harland Wood, and Feodor Lynen. J. Biol. Chem. 279,39187–39194

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The Role of the Acyl Carrier Protein in Fatty AcidSynthesis: the Work of P. Roy VagelosAcyl Carrier Protein. III. An Enoyl Hydrase Specific for Acyl Carrier ProteinThioesters(Majerus, P. W., Alberts, A. W., and Vagelos, P. R. (1965) J. Biol. Chem. 240, 618–621)

Acyl Carrier Protein. VII. The Primary Structure of the Substrate-binding Site(Majerus, P. W., Alberts, A. W., and Vagelos, P. R. (1965) J. Biol. Chem. 240, 4723–4726)

P. Roy Vagelos was born in Westfield, NJ in 1929. He received an A.B. degree from theUniversity of Pennsylvania in 1950 and an M.D. from Columbia University’s College ofPhysicians and Surgeons in 1954. Following an internship and residency at the MassachusettsGeneral Hospital in Boston, he joined the National Institutes of Health. There he launched acareer as a research scientist under the guidance of Earl Stadtman, who authored a previousJournal of Biological Chemistry (JBC) Classic (1). With Stadtman, Vagelos demonstrated thatlong-chain fatty acid synthesis is catalyzed by an enzyme complex in which malonyl-CoA is thesource of active acetate.

From 1956 to 1966, Vagelos served as Senior Surgeon and then Section Head of ComparativeBiochemistry in the National Heart Institute’s Laboratory of Biochemistry. During this time,he continued to study fatty acid synthesis, focusing on the role of acyl carrier protein (ACP).He discovered that the intermediates in fatty acid synthesis in Escherichia coli are linked toan acyl carrier protein via a thioester linkage. Vagelos published a series of papers on acylcarrier protein in the JBC, two of which are reprinted here as Classics.

During fatty acid synthesis, the acyl groups of acetyl-CoA and malonyl-CoA are initiallytransferred by acetyl and malonyl transacylases to the sulfhydryl group of ACP. Acetyl-ACPand malonyl-ACP are then condensed to form acetoacetyl-ACP, which is reduced to D(�)-�-hydroxybutyryl-ACP. The transacylases, condensing enzyme (acyl-malonyl-ACP condensingenzyme), and reductase (�-ketoacyl-ACP reductase) were characterized by Vagelos. This firstClassic focuses on the purification and properties of the enol hydrase (3-hydroxyacyl-ACPdehydratase) that catalyzes the dehydration of D(�)-�-hydroxybutyryl-ACP to crotonyl-S-ACP.

The second Classic deals with how substrates are linked to ACP. Vagelos had previouslyreported that, similar to CoA, substrates are bound to ACP via the sulfhydryl group of4�-phosphopantetheine. However, he noticed that despite this similarity between the twocarriers, thioesters of CoA could not substitute effectively for ACP in fatty acid synthesis. Uponfurther study of the structure of ACP, as reported in the second Classic, Vagelos discoveredthat 4�-phosphopantetheine is bound to ACP through a phosphodiester linkage to the hydroxylgroup of a serine residue.

In 1966, Vagelos assumed the chairmanship of the Department of Biological Chemistry atWashington University’s School of Medicine in St Louis, MO. He continued to work on fattyacid biosynthesis and metabolism and expanded his research to the synthesis of complex lipidsand the role of cholesterol in the biochemistry of the cell. In 1973 he became Director of theUniversity’s Division of Biology and Biomedical Sciences, which he founded. This Divisioneventually became a model for other universities. It included both the undergraduate Depart-ment of Biology and the Medical School in one umbrella unit, which was unheard of at thetime.

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Vagelos left academia in 1975 to join Merck Sharp & Dohme Research Laboratories as SeniorVice President for Research. In 1984 he was named an Executive Vice President of Merck and waselected to its Board of Directors, and in 1984 he became Merck’s Chief Executive Officer. Heserved as CEO and Chairman of the Board until 1994. Under his direction, the companyexpanded its philanthropic efforts as well as its pharmaceutical research. He is perhaps bestknown for his decision to make Merck’s Invermectin (Mectizan) available free to millions of peoplein Africa and Central America for the treatment of river blindness, a disease spread by black fliesthat causes chronic rashes, itching, weight loss, and blindness.

In recognition of his contributions to science, Vagelos received the American ChemicalSociety’s Enzyme Chemistry Award in 1967. He was elected to both the National Academy ofSciences and the American Academy of Arts and Sciences in 1972 and to the AmericanPhilosophical Society in 1993. In 1989 he received the Thomas Alva Edison Award from thenNew Jersey Governor Thomas Kean. He is currently Chairman of the Board of RegeneronPharmaceuticals, Inc. as well as a member of the Board of Directors of the PrudentialInsurance Company.1

Vagelos’ coauthors on several of the JBC acyl carrier protein papers, including the tworeprinted here, are Philip W. Majerus and Alfred W. Alberts. Majerus went on to become aProfessor at Washington University School of Medicine and has been a leader in phosphoi-nositide metabolism and signaling, platelet physiology, and blood coagulation. He is a memberof the National Academy of Sciences and has won numerous awards for his research, includingthe 1998 Bristol-Myers Squibb Award for Distinguished Achievement in Cardiovascular/Metabolic Research. Alberts moved from Washington University to Merck with Vagelos andwas the lead scientist in Merck’s development of the statin drugs Lovastatin and Zocor.


REFERENCES1. JBC Classics: Stadtman, E. R., and Barker, H. A. (1949) J. Biol. Chem. 180, 1085–1093; Mura, U., Chock, P. B.,

and Stadtman, E. R. (1981) J. Biol. Chem. 256, 13022–13029 (http://www.jbc.org/cgi/content/full/280/26/e23)2. Hawthorne, F. (2003) The Merck Druggernaut, John Wiley & Sons, Inc., Hoboken, NJ3. Park, B. S. The Stadtman Way: a Tale of Two Biochemists at NIH. http://history.nih.gov/exhibits/stadtman/

index.htm (An online exhibit produced by the Office of NIH History in collaboration with the National Heart,Lung, and Blood Institute)

1 All biographical information on P. Roy Vagelos was taken from Refs. 2 and 3.


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How Aspirin Interferes with Cyclooxygenase Activity:the Work of William L. SmithStimulation and Blockade of Prostaglandin Biosynthesis(Smith, W. L., and Lands, W. E. M. (1971) J. Biol. Chem. 246, 6700–6702)

The Aspirin and Heme-binding Sites of Ovine and Murine Prostaglandin Endoper-oxide Synthases(DeWitt, D. L., el-Harith, E. A., Kraemer, S. A., Andrews, M. J., Yao, E. F., Armstrong,R. L., and Smith, W. L. (1990) J. Biol. Chem. 265, 5192–5198)

William L. Smith, Jr. was born inTulsa, Oklahoma in 1945. He and hisfamily moved to the Chicago areawhen he was an infant and then toFort Collins, Colorado when he was inhigh school. After graduating fromhigh school, Smith enrolled in a pre-med program at the University of Col-orado in the fall of 1963, even thoughhe had no idea what sort of career hewanted to pursue. However, thischanged during the first couple yearsof college when he took several chem-istry courses that were taught by ex-cellent teachers. His admiration forthese professors led to his decision toattend graduate school. Originally, heplanned to go into physical organicchemistry but chose biological chem-istry when he was told that physicalorganic chemistry was already ahighly populated field. “Little did Iknow that the same thing wouldhappen in biochemistry by the time Iwas searching for a position,” recallsSmith.

After graduating with a B.A. in1967, Smith decided to attend theUniversity of Michigan and work withWilliam E. M. Lands. This decisionwas influenced by three papers by

Mats Hamberg and Bengt Samuelsson (1–3) published in the Journal of Biological Chemistry(JBC) that Lands gave him to read.“These papers dealt with the mechanism of oxygenation ofdihomo-�-linolenic acid and other fatty acids by soybean lipoxygenase and sheep seminalvesicle cyclooxygenase (i.e. prostaglandin H2 synthase 1 or PGHS-1),” recalls Smith. “In thecourse of these studies they labeled the �8 carbon (C-13) of the substrate with tritium in the

William L. Smith

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proS and proR orientations and found a distinct kinetic isotope effect in the removal of theproS hydrogen. This indicated that the rate determining step was abstraction of this hydrogenfrom the fatty acid. To this day, I find this to be a brilliant experiment and an exciting outcome.Almost 25 years later we provided evidence that Tyr-385 of PGHSs abstracted this hydrogenatom from the fatty acid (4).” More information on Samuelsson’s work on the prostaglandinscan be found in his JBC Classic (5).

Just as Smith was finishing up his thesis work with Lands, John Vane reported thatacetylsalicylic acid (aspirin) and another well known nonsteroidal anti-inflammatory drugcalled indomethacin blocked the biosynthesis of prostaglandins from arachidonic acid (6, 7).Smith had just set up an O2 electrode assay to measure prostaglandin production in acetonepowder preparations of sheep seminal vesicle microsomes, and he and Lands immediatelytested aspirin and indomethacin in this assay. As reported in the first JBC Classic reprintedhere, they found that aspirin and indomethacin blocked arachidonic acid-induced O2 uptakeand concluded that these drugs were blocking oxygenase activity. They also observed that thetwo drugs acted in a time-dependent manner, suggesting that they were causing a chemicalmodification of their target. Subsequently, it was shown that the acetyl group of aspirin wasincorporated into a protein (8) that Smith later purified (9) in his first JBC paper as anindependent scientist. The protein is now known as prostaglandin endoperoxide H synthase-1(PGHS-1) or cyclooxygenase-1 (COX-1).

Smith completed his Ph.D. work in under 4 years, and in 1971 went to the University ofCalifornia at Berkeley to work with Clinton E. Ballou. There he changed the focus of hisresearch and worked on polysaccharide structures. Although he enjoyed this work, he decidedhe was more interested in solving problems that he felt were more biomedically relevant. Healso realized that he still had an intense interest in prostaglandins. In 1974, Smith took a jobas a senior scientist at Mead Johnson in Evansville, Indiana where he spent his time studyingplatelet aggregation, prostaglandin formation, and arachidonic acid mobilization.

A year later, he accepted a position in the Department of Biochemistry at Michigan StateUniversity where he remained for 28 years and served as chair for the last 8 years of his timethere. At Michigan State, Smith continued to work on prostaglandins, focusing, among otherthings, on the acetylation of PGHS-1. Work in other laboratories suggested that aspirin wasacetylating a serine residue on the enzyme (10, 11). In 1988 Smith and his long time colleagueDavid DeWitt were able to clone and sequence PGHS-1 cDNA derived from seminal vesicles(12). They observed that the acetylated serine corresponded to Ser-530. Two years later, asreported in the second JBC Classic reprinted here, Smith and his colleagues showed thatsubstitution of Ser-530 with alanine rendered the protein refractory toward aspirin but hadrelatively little effect on the kinetic properties of the cyclooxygenase. They concluded that theSer-O-acetyl protrudes into the cyclooxygenase active site thereby interfering with arachidonicacid binding. This is the most definitive biochemical work on how aspirin works at themolecular level to interfere with cyclooxygenase activity. Smith later showed that substitu-tions of Ser-530 with bulkier residues such as threonine and asparagine gradually reducedcyclooxygenase activity (13).

In 2002 Smith decided to step down as Chair of Biochemistry at Michigan State, saying “Myopinion is that administrators should serve no longer than American presidents.” Shortlythereafter he was given the opportunity to become Chair of the Biological Chemistry Depart-ment at the University of Michigan. Still at the University of Michigan today, he continues towork on prostaglandins.

In recognition of his many contributions to science, Smith has received several awards andhonors. These include the 1991 Treadwell Award from George Washington University, the1992 Distinguished Faculty Award from Michigan State University, the 1996 Abraham WhiteDistinguished Scientific Achievement Award from George Washington University, the 1997Senior Aspirin Award from Bayer Corporation, the 1999 Michigan Universities Association ofGoverning Boards Award, the 2004 State of Michigan Scientist of the Year Award, the 2004Berzelius Lectureship from the Karolinska Institute, the 2004 Avanti Award from the Amer-ican Society for Biochemistry and Molecular Biology, the 2006 William C. Rose Award from theAmerican Society for Biochemistry and Molecular Biology, and the 2006 Hayaishi Lectureshipfrom Hamamatsu University.1

1 We thank William L. Smith for providing background information for this introduction.

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Smith’s co-author on the first Classic, William E. M. Lands, was a young faculty member atthe University of Michigan when Smith joined his laboratory. Lands subsequently left Mich-igan in 1980 to become Chair of Biochemistry at the University of Illinois, Chicago, and latermoved to the National Institutes of Health in 1990, where he served as the Senior ScientificAdvisor to the Director of the National Institute on Alcohol Abuse and Alcoholism. Lands is thediscoverer of the “retailoring” pathway for membrane phospholipid synthesis. He is an au-thority on essential fatty acids and is credited with recognizing the beneficial effects ofbalancing excess �-6 fatty acids with dietary �-3 fatty acids. In recognition of his work, theUniversity of Michigan’s Department of Biological Chemistry endowed a lectureship in nutri-tional biochemistry in honor of Lands.


REFERENCES1. Hamberg, M., and Samuelsson, B. (1967) On the specificity of the oxygenation of unsaturated fatty acids catalyzed

by soybean lipoxidase. J. Biol. Chem. 242, 5329–53352. Hamberg, M., and Samuelsson, B. (1967) On the mechanism of the biosynthesis of prostaglandins E1 and F1�.

J. Biol. Chem. 242, 5336–53433. Hamberg, M., and Samuelsson, B. (1967) Oxygenation of unsaturated fatty acids by the vesicular gland of sheep.

J. Biol. Chem. 242, 5344–53544. Shimokawa, T., Kulmacz, R. J., DeWitt, D. L., and Smith, W. L. (1990) Tyrosine 385 of prostaglandin endoperoxide

synthase is required for cyclooxygenase catalysis. J. Biol. Chem. 265, 20073–200765. JBC Classics: Bergstrom, S., Ryhage, R., Samuelsson, B., and Sjovall, J. (1963) J. Biol. Chem. 238, 3555–3564;

Hamberg, M., and Samuelsson, B. (1967) J. Biol. Chem. 242, 5336–5343 (http://www.jbc.org/cgi/content/full/281/9/e9)

6. Vane, J. R. (1971) Inhibition of prostaglandin synthesis as a mechanism of action for aspirin-like drugs. Nat. NewBiol. 231, 232–235

7. Ferreira, S. H., Moncada, S., and Vane, J. R. (1971) Indomethacin and aspirin abolish prostaglandin release fromthe spleen. Nat. New Biol. 231, 237–239

8. Roth, G. J., Stanford, N., and Majerus, P. W. (1975) Acetylation of prostaglandin synthase by aspirin. Proc. Natl.Acad. Sci. U. S. A. 72, 3073–3076

9. Hemler, M., Lands, W. E., and Smith, W. L. (1976) Purification of the cyclooxygenase that forms prostaglandins.Demonstration of two forms of iron in the holoenzyme. J. Biol. Chem. 251, 5575–5579

10. Van Der Ouderaa, F. J., Buytenhek, M., Nugteren, D. H., and Van Dorp, D. A. (1980) Acetylation of prostaglandinendoperoxide synthetase with acetylsalicylic acid. Eur. J. Biochem. 109, 1–8

11. Roth, G. J., Machuga, E. T., and Ozols, J. (1983) Isolation and covalent structure of the aspirin-modified,active-site region of prostaglandin synthetase. Biochemistry 22, 4672–4675

12. DeWitt, D. L., and Smith, W. L. (1988) Primary structure of prostaglandin G/H synthase from sheep vesiculargland determined from the complementary DNA sequence. Proc. Natl. Acad. Sci. U. S. A. 85, 1412–1416

13. Shimokawa, T., and Smith, W. L. (1992) Prostaglandin endoperoxide synthase. The aspirin acetylation region.J. Biol. Chem. 267, 12387–12392

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30 Years of Cholesterol Metabolism: the Work ofMichael Brown and Joseph GoldsteinBinding and Degradation of Low Density Lipoproteins by Cultured HumanFibroblasts. Comparison of Cells from a Normal Subject and from a Patientwith Homozygous Familial Hypercholesterolemia(Goldstein, J. L., and Brown, M. S. (1974) J. Biol. Chem. 249, 5153–5162)

Joseph Leonard Goldstein was born in 1940 in Sumter, South Carolina. He attendedWashington and Lee University in Lexington, Virginia and received a B.S. degree in chemistryin 1962. Goldstein then went to the Southwestern Medical School at the University of TexasHealth Science Center in Dallas where he was inspired to pursue a career in academicmedicine by the Chairman of the Department of Internal Medicine, Donald W. Seldin.

During Goldstein’s last year in medical school, Seldin offered him a future faculty appoint-ment if he agreed to specialize in genetics and return to Dallas to establish a division ofmedical genetics in the Department of Internal Medicine. Goldstein initially declined, andafter receiving his M.D. in 1966, he moved to Boston where he was an intern and resident atthe Massachusetts General Hospital. It was there that he first met and developed a friendshipwith Michael Stuart Brown, his long term scientific collaborator.

Brown, who was born in 1941 in Brooklyn, New York, graduated in 1962 from the Universityof Pennsylvania, with a major in chemistry. He received his M.D. degree from the Universityof Pennsylvania School of Medicine in 1966, after which he became an intern and resident atthe Massachusetts General Hospital.

After they completed their training in 1968, both Brown and Goldstein obtained positions at theNational Institutes of Health in Bethesda, Maryland. Brown joined the Laboratory of Biochem-istry at the National Heart, Lung, and Blood Institute (NHLBI). Goldstein worked with Nobellaureate Marshall W. Nirenberg and also worked as a clinical associate at NHLBI, serving asphysician to the patients of Donald S. Fredrickson, who was an expert on disorders of lipidmetabolism. Several of these patients had homozygous familial hypercholesterolemia, a conditionthat causes severe elevations in cholesterol levels. Goldstein discussed these patients extensivelywith Brown and, in view of their common interest in metabolic disease, convinced Brown to joinhim as a faculty member at the University of Texas Health Science Center at Dallas, where theywould work collaboratively on the genetic regulation of cholesterol metabolism.

In 1971 Brown joined the division of Gastroenterology in the Department of InternalMedicine at the University of Texas Southwestern Medical School. Before going back to Dallas,Goldstein spent 2 years with Arno G. Motulsky at the University of Washington in Seattle,studying human genetics. He returned to the University of Texas Health Science Center in1972 and was appointed Assistant Professor in the Department of Internal Medicine and headof the medical school’s first Division of Medical Genetics.

Together, Brown and Goldstein began to address the task of identifying the genetic defect infamilial hypercholesterolemia. They started by observing tissue cultures of fibroblasts har-vested from healthy individuals and individuals with familial hypercholesterolemia. They setup a microassay to measure the activity of 3-hydroxy-3-methylglutaryl coenzyme A reductase,the rate-determining enzyme of cholesterol biosynthesis, in the fibroblasts. Soon it becameclear that the cholesterol transport protein, low density lipoprotein (LDL), suppressed theactivity of 3-hydroxy-3-methylglutaryl coenzyme A reductase. Because high density lipopro-

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tein (HDL) was unable to do this, Brown and Goldstein suspected that a receptor might beinvolved in the control of 3-hydroxy-3-methylglutaryl coenzyme A reductase.

The existence of an LDL receptor was confirmed when Brown and Goldstein radiolabeled LDLwith 125I and incubated it with normal and familial hypercholesterolemial fibroblasts. As re-ported in the JBC Classic reprinted here, their studies showed that normal cells had high affinitybinding sites for 125I-LDL whereas familial hypercholesterolemial cells did not. Binding of LDLto the high affinity membrane receptor sites suppressed the synthesis of 3-hydroxy-3-methylglu-taryl coenzyme A reductase and also facilitated the degradation of LDL when it was present atlow concentrations. Cells from subjects with familial hypercholesterolemia not only lacked thebinding sites but were also resistant to suppression of 3-hydroxy-3-methylglutaryl coenzyme Areductase activity by LDL and were deficient in high affinity degradation of LDL.

However, the question of how LDL generated the signal that suppressed 3-hydroxy-3-methyl-glutaryl coenzyme A reductase still remained. The answer to this question came from studies ofsurface-bound 125I-LDL. Brown and Goldstein found that the receptor-bound LDL remained onthe cell’s surface for less than 10 min. Within this time most of the surface-bound LDL particlesentered the cell. Within another 60 min the protein component of 125I-LDL was digested com-pletely. The only cellular organelle that could have degraded LDL so completely and rapidly wasthe lysosome. This was eventually confirmed, and Brown and Goldstein also showed that thecholesterol that was generated from lysosomal degradation of LDL acted as the second messengerresponsible for suppressing 3-hydroxy-3-methylglutaryl coenzyme A reductase activity.

As is often the case with truly novel, groundbreaking discoveries, the work presented in thisJBC Classic was not met with great enthusiasm by journal reviewers. The initial review of thispaper by the JBC is presented in Fig. 1, and the decision letter from Associate Editor EugeneKennedy is shown in Fig. 2. This paper, the basis of the Nobel Prizes awarded to Brown andGoldstein, was eventually accepted.

By 1974 Brown and Goldstein had merged their laboratories. They continued their work onthe LDL receptor and eventually purified and sequenced it. In recognition of their work, theywere awarded the 1985 Nobel Prize in Physiology or Medicine “for their discoveries concerningthe regulation of cholesterol metabolism.”

Goldstein eventually became Associate Professor of Internal Medicine at the University ofTexas Southwestern Medical School (1974) and then Professor (1976). In 1977, he was madeChairman of the Department of Molecular Genetics at the University of Texas Health ScienceCenter and Paul J. Thomas Professor of Medicine and Genetics, a position that he still holdstoday. In 1985, he was named Regental Professor of the University of Texas, and in 1983 hebecame a Non-resident Fellow of The Salk Institute for Biological Sciences.

Fig. 1

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In 1974, Brown was promoted to the rank of Associate Professor of Internal Medicine at theUniversity of Texas Southwestern Medical School. He became a Professor in 1976. In 1977 hewas appointed Paul J. Thomas Professor of Medicine and Genetics and Director of the Centerfor Genetic Disease at the same institution. In 1985, Brown was named Regental Professor ofthe University of Texas.

In addition to the Nobel Prize, Brown and Goldstein’s work has been recognized by their receiptof numerous awards, including the Heinrich Wieland Prize for Research in Lipid Metabolism(1974), the American Chemical Society’s Pfizer Award for Enzyme Chemistry (1976), the PassanoAward (1978), the National Academy of Sciences’ Lounsbery Award (1979), the Gairdner Foun-dation International Award (1981), the Lita Annenberg Hazen Award (1982), the Association ofAmerican Medical Colleges’ Distinguished Research Award (1984), the American Heart Associ-ation’s Research Achievement Award (1984), the FASEB 3M Life Sciences Award (1985), theAlbert D. Lasker Award in Basic Medical Research (1985), the U. S. National Medal of Science(1988), the Albany Medical Prize in Biomedical Sciences (2003), and the Herbert Tabor/Journalof Biological Chemistry Lectureship (2005).

Both Brown and Goldstein were elected to the National Academy of Sciences and theAmerican Academy of Arts and Sciences. Goldstein is, or has been, a member of the editorialboard of several journals including the Annual Review of Genetics, Arteriosclerosis, Cell, theJournal of Biological Chemistry, the Journal of Clinical Investigation, and Science. Brown hasserved on the editorial boards of the Journal of Lipid Research, the Journal of Cell Biology,and Arteriosclerosis and Science.1,2


1 All biographical information on Joseph L. Goldstein was taken from Refs. 1 and 2.2 All biographical information on Michael S. Brown was taken from Refs. 2 and 3.

Fig. 2

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REFERENCES1. Goldstein, J. L., and Brown, M. S. (1993) A receptor-mediated pathway for cholesterol homeostasis. From Nobel

Lectures, Physiology or Medicine 1981–1990 (Frangsmyr, T., ed) World Scientific Publishing Co., Singapore2. Goldstein, J. L. (1986) Joseph L. Goldstein—Biography. In The Nobel Prizes 1985 (Odelberg, W., ed) Nobel

Foundation, Stockholm3. Brown, M. S. (1986) Michael S. Brown—Biography. In The Nobel Prizes 1985 (Odelberg, W., ed) Nobel Foundation,

Stockholm

Joseph L. Goldstein Michael S. Brown

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Salih Wakil’s Elucidation of the Animal Fatty AcidSynthetase Complex ArchitectureThe Architecture of the Animal Fatty Acid Synthetase. I. Proteolytic Dissection andPeptide Mapping(Mattick, J. S., Tsukamoto, Y., Nickless, J., and Wakil, S. J. (1983) J. Biol. Chem. 258,15291–15299)

The Architecture of the Animal Fatty Acid Synthetase. II. Separation of the Core andThioesterase Functions and Determination of the N-C Orientation of the Subunit(Mattick, J. S., Nickless, J., Mizugaki, M., Yang, C. Y., Uchiyama, S., and Wakil, S. J.(1983) J. Biol. Chem. 258, 15300–15304)

The Architecture of the Animal Fatty Acid Synthetase. III. Isolation and Character-ization of Beta-Ketoacyl Reductase(Wong, H., Mattick, J. S., and Wakil, S. J. (1983) J. Biol. Chem. 258, 15305–15311)

The Architecture of the Animal Fatty Acid Synthetase Complex. IV. Mapping ofActive Centers and Model for the Mechanism of Action(Tsukamoto, Y., Wong, H., Mattick, J. S., and Wakil, S. J. (1983) J. Biol. Chem. 258,15312–15322)

Salih Jawad Wakil was born in 1927 in Kerballa, Iraq. Because he placed third in the nationon the baccalaureate examination out of high school, he received a scholarship to the AmericanUniversity in Beirut. While at the American University he met Stanley Kerr, who introducedhim to biochemistry and gave him the opportunity to work in his laboratory. After graduatingin 1948, Wakil was accepted at the University of Washington, which he assumed was locatedin the U. S. capital. However, he arrived in the United States only to learn that he would haveto take a 3-day train journey from New York to his university in Washington State. In Seattle,Wakil worked with Donald Hanahan and finished his graduate studies in biochemistry in 31⁄2years. Next, he decided to do postdoctoral training at the Enzyme Institute of the Universityof Wisconsin, where he began to work on fatty acid oxidation. It was there that he helped toelucidate the steps by which fatty acids are oxidized and showed that fatty acids are synthe-sized and oxidized by different pathways.

Wakil was named assistant professor in 1956, but joined the Department of Biochemistry atthe Duke University School of Medicine in 1959 and rose to the rank of professor there (1965).At Duke, Wakil investigated fatty acid synthesis in Escherichia coli. He and Roy Vagelos, whowas featured in a previous Journal of Biological Chemistry (JBC) Classic (1), independentlystudied the role of acyl carrier protein as well as several of the individual reactions of fatty acidelongation. Wakil left Duke in 1971 to become professor and chairman of the Verna and MarrsMcLean Department of Biochemistry and Molecular Biology at Baylor College of Medicine inHouston,Texas. At Baylor, Wakil studied the multifunctional enzyme, fatty acid synthetase.The characterization of this enzyme complex is the subject of the four JBC Classics reprintedhere.

In vertebrates, the fatty acid synthetase complex exists as a dimer of what Wakil believedwere identical subunits derived from a single large mRNA. The complex contains the sevenenzymatic activities needed for the assembly of fatty acids: (i) acetyl transacylase, (ii) malonyltransacylase, (iii) �-ketoacyl synthetase, (iv) �-ketoacyl reductase, (v) �-hydroxyacyl dehy-

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dratase, (vi) enoyl reductase, and (vii) palmitoyl thioesterase, as well as an acyl carrier peptideto which the nascent chain is attached. These Classics, which were printed as a back-to-backseries in one issue of the JBC, present Wakil’s comprehensive proteolytic analysis of chickenfatty acid synthetase in which he assigned relative locations for the enzymatic activities in thecomplex.

In the first Classic, Wakil and his colleagues used seven different proteases to digest thesynthetase. They found that the sum of the molecular weights of each set of fragmentsgenerated by the proteases corresponded to the size of the synthetase subunit rather than thenative dimer, indicating that the synthetase was indeed a homodimer. The researchers alsoreported that the subunit is arranged into three major domains of Mr � 127,000, 107,000, and33,000.

Wakil describes the cleavage of chicken fatty acid synthetase by �-chymotrypsin in thesecond Classic. The complex was cleaved into two fragments. The larger 230-kDa fragmentcontained all the core activities involved in the assembly of the fatty acyl chain whereas thesmaller 33-kDa fragment retained the thioesterase activity which releases the completeproduct. Using amino acid sequence analysis, Wakil showed that the thioesterase domain islocated at the carboxyl terminus of the synthetase monomer.

In the third Classic, Wakil used trypsin and subtilisin to cleave fatty acid synthetase andisolated a polypeptide containing only �-ketoacyl reductase activity. Using a kallikrein/sub-tilisin double digestion, Wakil and his colleagues also isolated another fragment containing�-ketoacyl reductase activity as well as the phosphopantetheine prosthetic group. From this,Wakil concluded that the acyl carrier protein moiety is located in the 15-kDa segment thatseparates the �-ketoacyl reductase from the thioesterase domain.

In the fourth and final Classic, Wakil presents an architectural model for the synthetasebased on his results from the previous three papers. In Wakil’s model, domain I functions asa site for acetyl and malonyl substrate entry and acts as the site of carbon-carbon condensa-tion. Thus, this domain contains the amino terminus of the polypeptide and the �-ketoacylsynthetase and acetyl and malonyl transacylases. Domain II, the reductive domain, containsthe �-ketoacyl and enoyl reductases, probably the dehydratase, and the 4�-phosphopanteth-eine prosthetic group of the acyl carrier protein. Finally, domain III contains the thioesteraseactivity. Based on his observations, Wakil concluded that even though each subunit containsall the activities needed for fatty acyl synthesis, the actual synthesizing unit consists ofone-half of one subunit interacting with the complementary half of the other subunit. This isshown in the model in Fig. 2. Wakil, along with Bornali Chakravarty, Ziwei Gu, Subrah-manyam S. Chirala, and Florante A. Quiocho, subsequently solved the crystal structure of the

Salih J. Wakil

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thioesterase domain of human fatty acid synthetase (2). Recent crystallographic analysis ofboth the animal and fungal fatty acid synthases demonstrates that the structure is of ahead-to-head coiled dimer (3, 4).

Today, Wakil remains at Baylor where he is Distinguished Service Professor and BolinProfessor in the Department of Biochemistry and Molecular Biology. Most recently, his focushas been on acetyl-CoA carboxylase (ACC), which exists in two forms, ACC1 and ACC2. He hasdeveloped a transgenic mouse, which does not produce ACC2, and as a result can eat 20–30%more food and weighs 10% less than mice that produce the enzyme.

In honor of Wakil’s contributions to the field of fatty acid metabolism, he has received manyawards and honors. These include the Paul Lewis Award from the American Chemical Society(1967), the Chilton Award of the University of Texas Southwestern Medical Center (1985), theKuwait Prize of the Kuwait Foundation for the Advancement of Sciences (1988), the Yaman-ouchi USA Foundation Award (2001), and the Bristol-Myers Squibb Freedom to DiscoverAward (2005). In 1990, Wakil was the first Baylor College of Medicine faculty member to beelected to the National Academy of Sciences.


REFERENCES1. JBC Classics: Majerus, P. W., Alberts, A. W., and Vagelos, P. R. (1965) J. Biol. Chem. 240, 618–621; Majerus,

P. W., Alberts, A. W., and Vagelos, P. R. (1965) J. Biol. Chem. 240, 4723–4726 (http://www.jbc.org/cgi/content/full/280/35/e32)

2. Chakravarty, B., Gu, Z., Chirala, S. S., Wakil, S. J., and Quiocho, F. A. (2004) Human fatty acid synthase:structure and substrate selectivity of the thioesterase domain. Proc. Natl. Acad. Sci. U. S. A. 101, 15567–15572

3. Smith, S. (2006) Architectural options for a fatty acid synthase. Science 311, 1251–12524. Maier, T., Jenni, S., and Ban, N. (2006) Architecture of mammalian fatty acid synthase at 4.5 A resolution. Science

311, 1258–1262

Fig. 2

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N-Myristoyltransferase Substrate Selection and Catalysis:the Work of Jeffrey I. GordonMyristoyl CoA:Protein N-Myristoyltransferase Activities from Rat Liver and YeastPossess Overlapping Yet Distinct Peptide Substrate Specificities(Towler, D. A., Adams, S. P., Eubanks, S. R., Towery, D. S., Jackson-Machelski, E.,Glaser, L., and Gordon, J. I. (1988) J. Biol. Chem. 263, 1784–1790)

Isothermal Titration Calorimetric Studies of Saccharomyces cerevisiae Myristoyl-CoA:Protein N-Myristoyltransferase. Determinants of Binding Energy and CatalyticDiscrimination among Acyl-CoA and Peptide Ligands(Bhatnagar, R. S., Jackson-Machelski, E., McWherter, C. A., and Gordon, J. I. (1994)J. Biol. Chem. 269, 11045–11053)

Jeffrey I. Gordon obtained his A.B. fromOberlin College in 1969 and his M.D. fromthe University of Chicago in 1973. He thenbecame an intern and junior assistant resi-dent at Barnes Hospital in St. Louis beforespending 3 years as a research associate inthe Laboratory of Biochemistry at the Na-tional Cancer Institute (National Institutesof Health). Gordon returned to Barnes Hos-pital as a Senior Assistant Resident in 1978and was concurrently a Chief Medical Res-ident at John Cochran VA Hospital in St.Louis. He became an assistant professor atWashington University in St. Louis in 1981and has remained there ever since. He iscurrently Director of the Center for GenomeSciences as well as the Dr. Robert J. GlaserDistinguished University Professor.

Gordon is probably best known for hisresearch on gastrointestinal developmentand how gut bacteria affect normal intesti-nal function and predisposition to healthand to certain diseases. However, he hasalso made significant contributions to ourknowledge about myristoylation, the post-translational process by which a myristoylgroup is covalently attached via an amidebond to the �-amino group of an N-terminalglycine residue of a nascent polypeptide.

Some of Gordon’s work on N-myristoyltransferase, the enzyme responsible for myristoylation,is the subject of the two Journal of Biological Chemistry (JBC) Classics reprinted here.

Gordon started doing research on N-myristoyltransferase when Luis Glaser, a fellow scien-tist at Washington University, accepted a position at the University of Miami and transferred

Jeffrey I. Gordon

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the project, along with his student Dwight Towler, to Gordon’s laboratory. Glaser and Towlerhad already partially purified N-myristoyltransferase and were using peptides with alteredsequences to test as enzyme substrates in an in vitro assay system (1, 2). From their studiesthey had determined that residues occupying positions 1, 2, and 5 in peptide substrates (wherethe myristoylated glycine is residue 1) have important effects on protein-ligand interactions.In Gordon’s laboratory, Towler worked out a purification scheme for N-myristoyltransferasefrom yeast (3) and continued to investigate the primary structural characteristics of theenzyme’s peptide substrates. As reported in the first JBC Classic, Towler and Gordon screenedover 80 synthetic peptides and discovered that a substrate hexapeptide contains much of theinformation necessary for recognition by N-myristoyltransferase. They also identified a num-ber of potential N-myristoyl proteins from searches of available protein data bases.

In the second JBC Classic, Gordon and his colleagues report on the kinetics of the reactionin which N-myristoyltransferase transfers myristate from myristoyl-CoA to the amino-termi-nal glycine nitrogen of its substrate. Using isothermal titration calorimetry they quantified theeffects of varying acyl chain length and removing the 3�-phosphate group of CoA on theenergetics of interaction between N-myristoyltransferase and acyl-CoA ligands. From thesestudies, they were able to gain insights into how the enzyme selects its substrates and aboutits catalytic mechanism.

In recognition of his contributions to science, Gordon has received many awards and honors.These include the 1990 American Federation for Clinical Research Young Investigator Award,the 1990 National Institute of Diabetes and Digestive and Kidney Diseases Young ScientistAward, the 1992 American Gastroenterological Association (AGA) Distinguished AchievementAward, the 1994 Marion Merrell Dow Distinguished Prize in Gastrointestinal Physiology, the2003 Janssen/AGA Award for Sustained Achievement in Digestive Sciences, and the 2003Horace W. Davenport Distinguished Lectureship from the American Physiological Association.Gordon is also a member of the National Academy of Sciences and the American Academy ofArts and Sciences.

Luis Glaser, who was responsible for initiating Gordon’s investigations of N-myristoyltrans-ferase, has also led a fruitful career in science. Glaser, who grew up in Mexico, attended theUniversity of Toronto for his undergraduate education and earned his Ph.D. from WashingtonUniversity. After graduating, he joined the faculty of Washington University where he re-mained for the next 30 years, spending the last 10 years as Chairman of the Department ofBiology and Chemistry and Director of the Division of Biology and Biomedical Science. In 1986,Glaser accepted an offer to become Executive Vice President and Provost at the University ofMiami. He retired from that position in July of 2005 and is currently a Professor of Biology andSpecial Assistant to the President.


REFERENCES1. Towler, D., and Glaser, L. (1986) Protein fatty acid acylation: enzymatic synthesis of an N-myristoylglycyl peptide.

Proc. Natl. Acad. Sci. U. S. A. 83, 2812–28162. Towler, D. A., Eubanks, S. R., Towery, D. S., Adams, S. P., and Glaser, L. (1987) Amino-terminal processing of

proteins by N-myristoylation. Substrate specificity of N-myristoyltransferase. J. Biol. Chem. 262, 1030–10363. Towler, D. A., Adams, S. P., Eubanks, S. R., Towery, D. S., Jackson-Machelski, E., Glaser, L., and Gordon, J. I.

(1987) Purification and characterization of yeast myristoyl CoA:protein N-myristoyltransferase. Proc. Natl.Acad. Sci. U. S. A. 84, 2708–2712

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Hitler’s Gift and the Era of Biosynthesis

Published, JBC Papers in Press, September 14, 2001, DOI 10.1074/jbc.R100051200Eugene P. Kennedy‡From the Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School,Boston, Massachusetts 02115

Before the Second World War biochemistry in the United States had a strong flavor ofclinical chemistry. It was much occupied with problems of analysis of blood and tissues and thedetermination of the structures of body constituents. This was important and indeed essentialwork, but American students had to go abroad to Germany or to England for training in whatcame to be called dynamic aspects of biochemistry. After the war, the flow of students waslargely reversed. This transformation was in considerable part the result of new insights andnew approaches brought to America by immigrant scientists.

It is a remarkable fact that as late as 1945 when I began graduate studies in biochemistryat the University of Chicago almost nothing was known about the linked reactions leading tothe biosynthesis of any of the major types of cell constituents, carbohydrates, lipids, proteins,or nucleic acids. However, this picture was about to change with dramatic rapidity. The latterhalf of the 20th century became the era of biosynthesis. Now, in 2001, we know in great detailthe patterns of reactions leading to the formation of each of these classes of cellular materials,although to be sure much remains to be learned about the regulation and integration ofbiosynthetic processes in living organisms.

The achievements of three biochemists, Fritz Lipmann, Rudolf Schoenheimer, and KonradBloch, greatly stimulated this flowering of biosynthetic studies in the United States at themid-20th century. Each had been driven out of Germany by the brutal anti-Semitism of theNazi regime. Each was an important part of what has been called Hitler’s gift (1) to Americanand British science.

In helping to bring about the transition to the era of biosynthesis, Fritz Lipmann made clearthe crucial role of “energy-rich” phosphates in driving biosynthetic reactions and showed howthis principle operated in the formation of the much sought and highly elusive “active acetate”involved in so many pathways. Rudolf Schoenheimer helped put into the hands of biochemiststheir most subtle and versatile approach, that of the isotope tracer technique, and with its aidrevealed the dynamic state of body constituents. Konrad Bloch’s work on the formation ofcholesterol illustrated how the insights of Lipmann and Schoenheimer could be combined in amasterpiece of biochemistry to solve a problem of great medical as well as biological significance.

Fritz Lipmann: The Energetics of BiosynthesisFritz Lipmann (Fig. 1), who helped to shape the development of modern biochemistry, was

born in Koenigsberg, East Prussia in 1899 into a Jewish family of the professional class (2). In1917, he began the study of medicine. In 1918, while still a medical student, he was draftedinto the German army and spent the rest of the war in the medical corps in France. Releasedfrom the army, Lipmann resumed his medical studies and received the M.D. degree in 1921.He soon abandoned plans for the practice of medicine in favor of biochemical research, but healways valued the broad view of biology his medical education had given him, concluding: “The

‡ To whom correspondence may be addressed. E-mail: [email protected].

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biological education to which the observant student is exposed in medicine is a superiorpreparation for any career.” Indeed, the study of medicine offered the most comprehensiveview of biology then available. Many of the greatest figures in biochemistry early in the 20thcentury, including Warburg, Meyerhof, and Krebs, were trained as physicians. The breadth ofhis background helped give Lipmann the confidence that nothing in biology was beyond hisrange. Again and again, he proved ready to tackle new problems, no matter how far removedfrom previous work in his laboratory.

Turning to a career in research rather than the practice of medicine, Lipmann realized thatthe most fruitful approach to biological problems was through chemistry. He began a programleading to a Ph.D. in chemistry. His work for the dissertation, begun in 1927, was carried outin the laboratory of Otto Meyerhof. Meyerhof, whose work on glycolysis in muscle earned hima Nobel Prize, had a laboratory on the first floor of the Kaiser-Wilhelm Institute for Biology inBerlin, a city that was then the leading center of science in the world. Lipmann felt that hisexperience in Meyerhof’s laboratory was in many ways the origin of all his later work. His mostintense admiration, however, was reserved for Warburg. As Lipmann later recalled (3): “At thetop of everything, on the uppermost floor, was Otto Warburg. Warburg already had a mysteryabout him. We admired him boundlessly but saw little of him . . . ”

In Meyerhof’s laboratory, Lipmann worked on the role of creatine phosphate in musclecontraction. It was of course known that muscle contraction, with its attendant production oflactic acid, is intimately linked to glycolysis. The energetics of this linkage, however, remainedobscure. Lipmann (3) commented on “ . . . the vagueness of the understanding, then prevalent,of both the intermediary path of glycolysis and the mechanism of action of energy-richphosphate.” This work did much to turn Lipmann’s thinking to the role of phosphorylatedintermediates in energy transduction.

In 1930, Lipmann was already aware that a career for a Jewish scientist in Germany wasfraught with difficulty and peril. There began a period of Wanderjahre before he finally founda position that offered both independence and scope. However, he wished to remain in Berlinat least for a time to be near his fiancee Freda Hall (3). He became an assistant to AlbertFischer, working on problems of tissue culture. In 1931 during a hiatus in the work of Fischer’slaboratory caused by its move from Berlin to Copenhagen, Lipmann, newly married to FredaHall, traveled to the United States to work at the Rockefeller Institute in New York in thelaboratory of Phoebus A. Levene on the biochemistry of phosphoproteins. Here he succeeded inisolating phosphoserine from partial acid hydrolysates of egg phosphoprotein.

FIG. 1. Fritz Lipmann. Photo by John Brook, made available through the courtesy of Ms. Freda Hall Lipmann.

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In 1932, Lipmann rejoined Fischer’s group in its new quarters in the Carlsberg Laboratoriesin Copenhagen, where he was to remain until 1939. He was free to work independently inpursuit of his own ideas. At about this time, Otto Warburg was making his great discoverieselucidating the central mechanisms of glycolysis. Why the splitting of glucose involved phos-phorylated intermediates had long been a great puzzle, which Warburg now solved.

In 1905 Arthur Harden, working in London, had discovered that glycolysis requires aheat-stable, organic cofactor, which he termed “cozymase.” This cofactor proved to be remark-ably elusive. In 1929, more than two decades later, Hans von Euler received a Nobel Prize forhis work on its isolation and characterization, but it is clear from his Nobel lecture that he hadat that time no real idea of its true structure and function. It was Warburg and his collabo-rators (4) who isolated “cozymase” and showed that it contains a pyridine ring that undergoesalternate reduction and re-oxidation. It is of course the famous coenzyme NAD now known tofunction in many hundreds of enzyme-catalyzed redox reactions. Warburg also discovered thatthe oxidation of 3-phosphoglyceraldehyde by NAD is linked to the uptake of orthophosphateand the formation of 1,3-diphosphoglyceric acid. This acyl phosphate may then react with ADPto form ATP. For the first time, the bioenergetic function of glycolysis became clear. A portionof the free energy released during the breakdown of glucose is made available to the cell asATP.

Lipmann followed these developments closely and they deeply influenced his thinking. In1939, he turned to an investigation of the role of phosphate in the oxidation of pyruvate inextracts of the organism then called Bacterium acidificans longissimum (Delbrueckii). Hediscovered (5) that the oxidation of pyruvate was coupled to the uptake of orthophosphate andthe phosphorylation of AMP (presumably with the formation of ATP).

By analogy with the role of 1,3-diphosphoglyceric acid in glycolysis, he formulated thefollowing reactions.

Pyruvate � phosphate ¡ acetyl phosphate � 2�H]

Acetyl phosphate � AMP ¡ “adenosine polyphosphate”

REACTIONS 1 AND 2

The isolation from these enzyme preparations of highly labile acetyl phosphate present only invery small amounts was really not feasible with the methods then available. Lipmann neatlygot around this difficulty by synthesizing acetyl phosphate from acetyl chloride and trisilverphosphate. He then showed that this synthetic compound, like the presumed intermediate,was effectively utilized for the formation of “adenosine polyphosphate” in these extracts.(Much later, when I worked in Lipmann’s laboratory and read his early papers, I was greatlytaken by this strategy. I learned from it that it is sometimes easier to synthesize a suspectedintermediate in an enzyme system than to isolate it, a lesson that led me to synthesizeCDP-choline first and then demonstrate its role as coenzyme.)

The work on acetyl phosphate marked the beginning of Lipmann’s long and productiveengagement with both the role of phosphate esters in energy transduction and the problem of“active acetate.” In July of 1939, with the Nazi menace ever more threatening, Fritz and FredaLipmann left Copenhagen for the United States. There followed a difficult period in which hesought without success a position that would offer him security and scope commensurate withhis talents. In 1940, he was invited to present a talk in a symposium at the University ofWisconsin in Madison attended by many of the leading figures in American biochemistry.Lipmann, never a facile or polished speaker, vastly underestimated the time needed for thematerial he wished to present and finally, midway through his discourse, had to be interruptedby the chairman of the session (3). Later he felt that this painful episode was one of the factorsthat made it difficult for him to secure a suitable position.

In 1940, Lipmann was invited by F. F. Nord to contribute a chapter to the first volume of theseries, Advances in Enzymology. As Lipmann later (3) wrote: “ . . . I was happy when heaccepted my suggestion that I write about the role of phosphate bonds as carriers in energytransformations and in biosynthesis. This had begun to impress me as an extension of myexperience with acetyl phosphate. Some of the propositions made in that article must havebeen more novel than I realized.”

Now, in 2001, it is very difficult to realize the impact of this article (6), particularly onAmerican biochemists who had not closely followed the work in European laboratories.


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Lipmann clearly distinguished between two classes of phosphate compounds in living cells.The first class, phosphate esters of alcohols such as glycerophosphate with a free energy ofhydrolysis of 2–4 kcal, was termed by Lipmann as “energy-poor” phosphates, designated in theshorthand which he introduced as (�ph). These were to be sharply distinguished from anotherclass comprising pyrophosphates, acyl phosphates, enol phosphates, and nitrogen-linked phos-phates such as phosphocreatine. The free energy of hydrolysis of phosphates of this class is ofthe order of 8–12 kcal. In Lipmann’s terminology these are energy-rich phosphate bonds,designated with a symbol that was to become famous as the “wiggle bond” (�ph).

A great generalization was stressed in his essay. Photosynthesis and the breakdown oforganic foodstuffs provide energy to living cells, some part of which is captured in useful formas “energy-rich” phosphates, leading to the formation of ATP. Lipmann pointed out: “Indica-tions are found that the phosphate current can be utilized to carry out mechanical work . . .(and) to synthesize protoplasmic material as lecithin, nucleic acid, and so forth.” Lipmannmade it clear that the energy needed to drive biosynthetic processes must come from ATPeither directly or, as was soon to be found, indirectly. Before this time biosynthetic processescould be studied only in intact animals or in preparations such as tissue slices (developed byWarburg) in which cellular structure remained intact. The principal conceptual barrier to thestudy of cell-free systems was now removed. Biochemists began to add ATP (of varying degreesof purity!) to their enzyme systems when searching for biosynthetic reactions.

The 1941 essay is revealing in many ways of Lipmann’s style, which had a personal flavoreven when dealing with chemical thermodynamics. These were problems about which he hadthought deeply, and he conveyed his ideas in striking and forceful metaphors. Thus he spokeof a “phosphate potential” in analogy to an electrical potential and a “phosphate current” thatconveys energy as “energy-rich” phosphates are hydrolyzed. Critics pointed out that his use ofthe term “bond energy” to denote the energy released in breaking a bond was the opposite ofthe conventional use to denote the energy of bond formation, but Lipmann (3) tended to waveaside such criticism. “The physical chemist remains aloof. He may be forced to accept theusage, but he usually refrains from referring to the dilettante who originated it.”

Lipmann’s search for a suitable position now found a happy outcome in a rather unusualway. In 1941 Dr. Oliver Cope offered him an appointment in the Department of Surgery at theMassachusetts General Hospital. Although the space made available was at first quite limited,he was given complete freedom to follow his own ideas. Lipmann’s years at the MassachusettsGeneral Hospital were highly productive and led him to a Nobel Prize in 1953.

In 1941 the identity of “active acetate,” also described as the “two-carbon unit,” was one ofthe most pressing problems in intermediary metabolism. A growing body of evidence suggestedthat “active acetate” was the fundamental building block for the synthesis of sterols and fattyacids. Derived from the oxidation of pyruvate or of fatty acids, it could also react withoxalacetate to form citrate and thus enter the Krebs cycle for the final common pathway ofoxidative metabolism. Strongly encouraged by his success in identifying acetyl phosphate asan intermediate in the bacterial oxidation of pyruvate, Lipmann set out to examine its possiblerole as the elusive “active acetate” in animal tissues.

He chose to study the acetylation of sulfanilamide, known to occur in liver, because of theease with which this aromatic amine could be diazotized and coupled with a chromogen to forman intensely colored dye. The conversion of sulfanilamide to the unreactive N-acetyl derivativecould thus be easily measured. He succeeded in obtaining preparations from pigeon liver thatactively acetylated sulfanilamide but to his considerable disappointment found that acetylphosphate did not stimulate acetylation but instead was rapidly hydrolyzed (7). Significantly,however, he found that ATP as well as acetate was required for acetylation and furtherreported that enzyme preparations, inactivated by storage overnight at 7 °C, could be restoredto activity by the addition of boiled liver extract.

Nachmansohn and Machado had previously described a cofactor needed for the acetylationof choline. With the arrival of Kaplan in his laboratory, Lipmann’s cofactor was purified about100-fold and shown to be active also in the acetylation of choline (8). It appeared to be a generalcoenzyme for acetylation and hence the designation coenzyme A or CoA. The next step was thediscovery (9) in 1947 that CoA, by then purified about 700-fold, contains the vitamin panto-thenic acid. This was a very great advance.

A little later, in 1950, recommended by H. A. Barker, I entered Lipmann’s laboratory as apostdoctoral fellow following the footsteps of Earl Stadtman, who had just departed to take up


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a position at the National Institutes of Health. Stadtman had also come to Lipmann fromBarker’s laboratory. Lipmann’s group at this time included David Novelli, John Gregory,Morris Soodak, Harold Klein, Charles Du Toit, and Lipmann’s research assistant, Ruth Flynn.We were crammed into a single, tiny laboratory in the Massachusetts General Hospital nextto the famous Ether Dome, scene of the first (or so it was claimed) use of diethyl ether as ananesthetic. In the course of the year we were to move into spacious, even rather elegant,quarters in a newly constructed research building.

With abundant hair just turning gray and usually wearing a soft bow tie and a dark blueshirt, Lipmann presented a figure closer to that expected of an artist rather than a scientist.He spoke softly, and his sentences often trailed off into the distance. Lipmann’s mannertoward those who worked in his laboratory was rather formal. He was friendly but a littlealoof. He inspired nevertheless not only loyalty and admiration but also lasting affection inthose who worked under his direction.

At this time, Lipmann’s chief goal was the final purification of coenzyme A, which wasproving very difficult, and the determination of the structure of “active acetate,” the interme-diate with so many crucial roles in metabolism. Because acetyl phosphate, shown to be anactivated form of acetate in bacteria, was so labile, we surmised that acetyl-CoA, whatever itsstructure might be, would be even more labile, and this supposed lability was assumed toexplain the failure of our efforts to isolate it.

One day in 1951, I came upon an article in Angewandte Chemie (10) from the laboratory ofFeodor Lynen. He and his student Ernestine Reichert reported evidence for an essentialsulfhydryl residue in CoA. They had isolated acetyl-CoA and proved it to be a thioester! Ibrought the article at once to Lipmann who had not learned previously of this development. Hewas generous in praise of the work although Lynen had stolen some of his thunder. He wasparticularly impressed by the fact that in isolating acetyl-CoA from yeast, they had begun byboiling the yeast. We should have realized, Lipmann pointed out, that an intermediate thatplays such varied roles is unlikely to be so extremely labile as we had feared. Lipmann alsonoted that thioesters must be added to the list of biologically active “energy-rich” compounds.“Yes,” he mused in a discussion at this time, “there is a world of sulfur, like the world ofphosphorus, only smaller!”

In 1953 Lipmann shared the Nobel Prize with H. A. Krebs. Although the citation for theprize emphasized his work on CoA, Lipmann placed greater stress on his contributions tobioenergetics. “In my own judgment,” he wrote (3), “there was greater scope in the recognitionthat �P, as I had dubbed it, was acting as a biological energy quantum, carrying energypackages to metabolic function and biosynthesis.”

In 1957, he moved to the Rockefeller Institute. He continued to be remarkably productive ina wide variety of biosynthetic problems, further developing his grand themes of group activa-tion and the energetics of biosynthesis until his death in 1986 at the age of eighty-seven.

Rudolf Schoenheimer and the Dynamic State of Body ConstituentsThe single most important technical advance that transformed biochemistry in the 20th

century was the isotope tracer technique. Without it, the rapid growth of our knowledge ofbiosynthesis would be simply inconceivable. Georg Hevesy was the first to explore the biolog-ical usefulness of radioactive tracers in studies of the uptake of radiolead and its movementinto tissues of plants (11). It is to Rudolf Schoenheimer (Fig. 2), however, that we owe thebrilliant exploitation of the concept of isotopic tagging, that is the introduction of isotopes intospecific positions of organic molecules, whose metabolic transformations could then be traced.

Valuable accounts of Schoenheimer’s career have been published by Kohler (12) and byYoung and Ajami (13). He was born in Berlin in 1898 (12). Like Lipmann, he studied medicineand received the M.D. degree from the University of Berlin in 1922. Again like Lipmann, herecognized the need for deeper knowledge of chemistry and spent 3 years in the laboratory ofKarl Thomas in Leipzig, working largely on problems such as the chemical synthesis ofpeptides.

In 1926, Schoenheimer went to the Institute of Pathological Anatomy in Freiburg asassistant to Ludwig Aschoff, a leading expert on atherosclerosis (12). Schoenheimer began aninvestigation on the deposition of cholesterol into the arteries of rabbits fed a high level ofcholesterol in the diet. He was to pursue his interests in cholesterol metabolism for the rest ofhis life.


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It was here in Freiburg in 1930 that Schoenheimer encountered Hevesy, who wished tostudy the partition of labeled lead between normal and tumor tissue (12). Realizing hisinadequate background in biology, Hevesy asked Aschoff to suggest a collaborator for thiswork. Aschoff suggested Schoenheimer. Later, Hevesy (14) wrote: “It was in the course of theseinvestigations that Schoenheimer became familiar with the method of isotopic indicators,which he applied several years later with such great success . . . Never were more beautifulinvestigations carried out with isotopic indicators than those of the late Professor Schoenhei-mer . . . ”

Although the collaboration with Hevesy was undoubtedly significant for Schoenheimer’sthinking, his development of the use of isotopes was to go far beyond the scope of Hevesy’sapproach. In 1933 Schoenheimer, like so many others, was forced to leave Germany. TheJosiah Macy Foundation in the United States had begun in 1931 to support Schoenheimer’sresearch, and the director of the foundation, Ludwig Kast, now arranged an appointment forSchoenheimer in the Department of Biological Chemistry at Columbia, with salary andresearch funds supplied by the Foundation (12).

Hans T. Clarke, an organic chemist by training, had assumed the direction of the Depart-ment of Biological Chemistry in 1928, and he proceeded to make it the finest department in theUnited States. In an account of his career (15), Clarke stated: “Among the many benefits whichaccrued to Columbia University from the racial policy adopted by the Germans under theThird Reich was the arrival in our laboratory of various European-trained biochemists,notably Erwin Chargaff, Zacharias Dische, Karl Meyer, Rudolf Schoenheimer and HeinrichWaelsch. Erwin Brand, who joined our group during the same period, reached this countrysomewhat earlier. The scientific achievements subsequently made by these men are so wellknown that their enumeration is unnecessary.” Clarke modestly omitted to mention that hisown vision and humane instincts in welcoming these gifted refugees were by no means to befound in every American academic institution.

FIG. 2. Rudolf Schoenheimer. From Ref. 13 with permission. Photo made available through the courtesy of Mrs.Peter Klein.


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In 1932, also at Columbia University in the Department of Chemistry, Harold Urey discov-ered deuterium, the heavy isotope of hydrogen, by demonstrating the presence of new bandsin the positions predicted for a form of hydrogen of mass 2, in the spectrum of a sample ofhydrogen enriched in the heavier isotope by fractional distillation of liquid hydrogen. In 1934,Urey received a Nobel Prize for this work. Because separation of the isotopes of an element isa function of the ratio of their masses, isotopes of the heavier elements are very difficult toseparate. Deuterium, however, has twice the mass of ordinary hydrogen, and its preparationin pure form or as D2O (immediately dubbed “heavy water”) is comparatively straightforwardand was very soon undertaken in the laboratories of Urey and G. N. Lewis among many others.

The discovery of a completely new form of a substance of such universal importance as waterimmediately attracted great public interest all over the world. When Urey received his NobelPrize in 1934, Palmer, in his laudatory introduction of Urey, mentioned that large amounts ofheavy water were already being produced by an electrolytic process at the Norsk HydroConcern in Norway at the rate of about a half-liter per day (16). In 1940 after a more sinisteruse of heavy water as the moderator for atomic piles had emerged, this Norwegian heavy waterproduction facility was taken over by the German army of occupation. It then became thetarget for heroic and tragic efforts of Norwegian patriot saboteurs and the allied air forces todestroy it. The Germans finally dismantled it in 1945. The first biological experiments withD2O were relatively crude. For example, Lewis (17) reported that tobacco seeds suspended inpure D2O failed to germinate, and flatworms died when placed in water containing more than90% D2O. In these and other early experiments, the emphasis was on replacement of H2O asa medium for growth by D2O and not on the specific replacement of hydrogen by deuterium inmolecules of biological importance.

Urey, a physical chemist, stated that he was a biologist at heart. Indeed, at a later stage ofhis career at the University of Chicago he turned to fundamental biological research. With hisgifted collaborator Stanley Miller, he designed experiments that demonstrated the readysynthesis (under conditions that simulated the atmosphere of the early earth) of moleculesthat might plausibly be considered to be building blocks for the formation of cell substances.These studies greatly influenced many later investigations of the origin of life.

To promote the applications of the deuterium isotope to biological research, Urey persuadedWarren Weaver, head of the Rockefeller Foundation, to provide funds to permit David Rit-tenberg, a recent Ph.D. in physical chemistry in Urey’s department, to come to the Departmentof Biological Chemistry (12). As Hans Clarke commented (15): “In 1934, Schoenheimer madea new contact which proved to exert a fundamental influence on the nature of his work . . .David Rittenberg came from Urey’s group to the laboratory in which Schoenheimer had beenworking for a year. From their association there developed the idea of employing a stableisotope as a label in organic compounds, destined for experiments in intermediary metabolism,which should be biochemically indistinguishable from their natural analogs . . . ”

This new conception of Schoenheimer and his collaborators was a far cry from the simplemeasurement of the movement of a radioactive ion from one part of a plant or animal toanother, as had been done by Hevesy. In the new approach, the fate of the molecule into whichthe isotope had been incorporated was studied, not simply the isotope itself. Perhaps thenearest intellectual predecessor of this idea was the approach of Knoop, who in 1904 “labeled”fatty acids by the attachment of a phenyl residue to the �-carbon atom. Knoop found that if thefatty acid had an even number of carbon atoms, phenylacetic acid (linked to glycine in aso-called detoxification reaction) was recovered from the urine of dogs to which it had been fed.If on the other hand, the fatty acid had an odd number of carbon atoms, benzoic acid wassimilarly recovered. Knoop concluded that the phenyl residue could not be cleaved from the �carbon to which it was linked and more significantly correctly concluded that fatty acidoxidation in animal tissues must involve oxidation at the �-position. This result stronglyinfluenced later studies of fatty acid oxidation, but the work was subject to the objections thatphenyl-substituted fatty acids are very different from natural fatty acids, and a more seriouslimitation was that this type of labeling was not generally suitable for substances other thanfatty acids.

Schoenheimer was well aware of Knoop’s work. In a brief review in 1935 (18), Schoenheimerand Rittenberg pointed out: “Many attempts have been made to label physiological substancesby the introduction of easily detectable groups such as halogens and benzene nuclei. However,the physical and chemical properties of the resulting compounds differ so markedly from those


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of their natural analogs that they are treated differently by the organism. The interpretationof metabolic experiments involving such substances is therefore strictly limited. We havefound the hydrogen isotope deuterium to be a valuable indicator for this purpose . . . We haveprepared several physiological compounds (fatty acids and sterol derivatives) containing one ormore deuterium atoms linked to carbon, as in methyl or methylene groups . . . The number ofpossible applications of this method appear to be almost unlimited.”

At this period, mass spectrometers were still rare and finicky instruments. It was anadvantage of these early experiments that the content of deuterium in organic compoundscould be determined comparatively simply by combustion of the compound and very precisemeasurement of the density of the water so produced.

In 1935, it was a widely held doctrine that the bodily constituents of an adult animal werequite stable, while foodstuffs in the diet were immediately metabolized to provide energy andthe end products excreted. In their earliest experiments, Schoenheimer and Rittenberg foundevidence to overturn this doctrine. When fatty acids labeled with deuterium were fed to mice,most of the deuterated fat was first deposited in the fat depots. The fat burned in the body wasnot taken directly from the diet but from adipose tissue. Schoenheimer (19) concluded: “Thesefirst experiments with isotopes showed that the fats of the depots are not inert storagematerials but are constantly involved in metabolic reactions.”

To study the synthesis of fatty acids, Bernhard and Schoenheimer (20) administered D2O tomice and later measured the isotope content of their fatty acids. The saturated fatty acids werefound to contain relatively high levels of deuterium, but the polyunsaturated linoleic andlinolenic acids, known to be essential components of the diet, contained only traces. Theyconcluded that the mice carried out a very active de novo synthesis of saturated but not ofessential fatty acids. Because the total fat content of the mice did not change, the resultsindicated a rapid breakdown of body fats, equal to the rate of synthesis.

As might be expected, an important objective of Schoenheimer’s new program was aninvestigation of the metabolism of cholesterol. When cholesterol was isolated from mice givenD2O, Rittenberg and Schoenheimer (21) found from the rate of incorporation of deuterium intoit that cholesterol must be continually renewed with a half-time of the order of 3 weeks. Toaccount for the extensive incorporation of stably bound deuterium into the cholesterol mole-cule, it was concluded that its synthesis, like that of fatty acids, must involve the condensationof many small molecules.

A major extension of the range Schoenheimer’s investigations came with the concentrationof the isotope 15N by Urey and his collaborators in 1937. It was immediately applied to studiesof the metabolism of amino acids and proteins. In 1938, Schoenheimer et al. (22) reported thefirst experiments in which an amino acid in the diet, tyrosine, was labeled with 15N. “Theoriginal aim of this exploratory experiment was merely to find out whether in nitrogenequilibrium, the nitrogen in the urine is derived from the food proteins directly, or whetherdietary nitrogen is deposited, with liberation of an equivalent amount of tissue nitrogen forexcretion . . . The results indicate that in our rat the nitrogen of at least one amino acid,tyrosine, was only partly excreted in the urine, while almost half of it was retained in the bodyproteins.”

Here was another blow at the doctrine that ingested foods were immediately metabolizedand the products promptly excreted. Schoenheimer now found this view very naıve. If one putsa penny into a gumball machine, he asked, and a gumball comes out, does the machine turncopper into gum?

Schoenheimer had now become the central figure in Clarke’s Department of BiologicalChemistry. New and larger laboratory facilities were made available for him. His enthusiasmand vision attracted collaborators and students. As Kohler (12) has pointed out, he had becomethe leader of perhaps the first multidisciplinary biochemical laboratory. A physicist wasneeded for the preparation and measurement of isotopes. An organic chemist was employed forthe synthesis of isotopically labeled compounds, because of course none were available com-mercially. Biochemists were required for the separation and analysis of cell constituents.Technicians for animal care were also needed. Schoenheimer’s background in chemistry aswell as in biology and medicine made him especially effective in the leadership of thisdisparate group.

Schoenheimer’s investigations of protein metabolism, carried out with amino acids contain-ing 15N in the amino group and deuterium on the carbon chains provided results that had the


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greatest impact on biochemical thought. Briefly summarized (19), body proteins were found tobe in a state of continuous turnover. “The peptide bonds have to be considered as essentialparts of the proteins and one may conclude that they are rapidly and continually opened andclosed in the proteins of normal animals. The experiments give no direct indication as towhether the rupture is complete or partial.” The work thus raised questions that were tochallenge the next generation of biochemists.

Together with the earlier work on fat metabolism, a new and remarkable picture of theoverall metabolism of animals emerged. Schoenheimer summarized his conclusions (19): “Thelarge and complex molecules and their component units, fatty acids, amino acids, and nucleicacids, are constantly involved in rapid chemical reactions. Ester, peptide, and other linkagesopen; the fragments thereby liberated merge with those derived from other large moleculesand with those absorbed from the intestinal tract to form a metabolic pool of componentsindistinguishable as to origin . . . This idea can scarcely be reconciled with the classicalcomparison of a living being to a combustion engine nor with the theory of independentexogenous and endogenous types of metabolism . . . The classical picture must thus be replacedby one which takes account of the dynamic state of body structure.”

In 1941, Schoenheimer was invited to give the prestigious Dunham Lectures at the HarvardMedical School. The materials and notes that he prepared for the lectures, from which someof the quotations above are taken, were later published (19) under the title “The DynamicState of Body Constituents.” This lucid summary of his innovative work made a deep impres-sion on the biochemists of the generation to follow.

Schoenheimer had apparently been subject to attacks of depression and was undergoing aperiod of considerable personal stress when tragically in September of 1941 he ended his ownlife (12). Forty-three years of age at the time of his death, he was at the height of his powers.Fortunately many of the projects that he had begun were carried forward by very ablecollaborators, one whom took up the cholesterol problem.

Konrad Bloch and the Biosynthesis of CholesterolKonrad Bloch (Fig. 3) was born in 1902 in Neisse, a town in the eastern German province of

Silesia, the second child of a prosperous Jewish family (23). In his boyhood, Bloch evinced littleinterest in science other than nature studies, but his attendance in a course of organicchemistry at the Munich Technische Hochschule taught by Hans Fischer marked a turningpoint for him. Fischer, later to receive a Nobel Prize, was one of the remarkable group of giftedGerman chemists who then dominated the study of natural products. Although Fischer’slectures were delivered in a monotone, Bloch found the material fascinating and he realizedthat he had found his field (23).

In 1934, the brutal Nazification of Germany prevented Bloch from continuing his studiesthere. Hans Fischer came to his rescue by recommending his appointment at the Schweiz-erisches Hoehensforschungs Institut in Davos, Switzerland, the scene where Thomas Mannplaced the tuberculosis sanitarium in his novel The Magic Mountain.

In Davos, Bloch worked for a time on the lipids of the tubercle bacillus. In 1936, however, hewas refused permission to continue to reside in Switzerland. Desperate, he applied to R. J.Anderson at Yale, with whom he had some correspondence concerning his research. Hepromptly received two letters, the first from the Dean of the Medical School of Yale Universityinforming him that he had been appointed assistant in Biological Chemistry and the secondfrom Anderson informing him that there was no salary attached to this position. He showedthe first letter, but not the second, to the United States consul in Frankfurt and received alife-saving visa to immigrate to the United States.

Upon arrival in New York, Bloch applied to Hans Clarke’s department for admission as agraduate student. The sole formality in those happy days was an interview with Clarkehimself. The most important question, Bloch later jested, was: “Do you play a musicalinstrument?” Fortunately, Bloch could say that he played the cello, an answer agreeable toClarke, who loved chamber music.

Shortly after completion of his work for the Ph.D. degree under Clarke’s supervision, Blochjoined Schoenheimer’s group. In 1940, Schoenheimer suggested that he investigate the originof the hydroxyl oxygen in cholesterol. Was it water or O2? The thought that it might bemolecular oxygen showed the remarkable prescience of Schoenheimer because direct oxygen-ation was without precedent at that time. Unfortunately, Bloch found the technical problems


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of the mass spectrometry of oxygen compounds intractable in the state of technology of 1940and was forced to give up the project. In 1956, however, he returned to the problem and withhis student Tchen (24) showed that molecular oxygen is indeed the source of the hydroxyloxygen.

As Bloch (23) recalled: “Schoenheimer’s untimely death in 1941 left his associates withoutthe leader and the inspired leadership they so admired. We feared that we might have to lookfor jobs elsewhere, but Hans Clarke encouraged us to continue as heirs to the wealth of projectsSchoenheimer had begun and developed . . . How the division of ‘spoils’ came about I do notrecall—it may have been by drawing lots. At any rate, David Shemin ’drew’ amino acidmetabolism, which led to his classic work on heme biosynthesis. David Rittenberg was tocontinue his interest in protein synthesis and turnover, and lipids were to be my territory.”

Bloch now began his independent studies of the biosynthesis of cholesterol. It was aformidable enterprise. In the era before NMR, infrared, and mass spectroscopy, the determi-nation even of the chemical structure of cholesterol, with its 27 carbon atoms arranged in fourrings and with a branched hydrocarbon side chain, had been a challenge to the world’s greatestchemists of natural products. The Nobel Prizes in chemistry for 1927 and 1928 had beenawarded to Heinrich Wieland and Adolf Windaus, respectively, for their work on the structureof cholesterol and the closely related bile acids, but it was not until 1932 that the fully correctstructure was established.

In his 1928 Nobel lecture (25), Windaus stated: “This formula [of cholesterol] is verycomplicated and has no similarity to the formulae of sugars, fatty acids, or the amino acidswhich occur in protein. The synthesis of such a substance appears to the chemist particularlydifficult, and up to now I have not dared to attempt it, as success is extremely improbable.Furthermore, the majority of physiologists have not been inclined to believe the animalorganism capable of such a synthesis, for it is known that other seemingly simpler syntheses—e.g. that of tyrosine and tryptophane—have not succeeded in the animal organism.”

FIG. 3. Konrad Bloch. Photo made available through the courtesy of Mrs. Konrad Bloch.


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Bloch of course knew that Windaus’ pessimistic view of the capabilities of the animalorganism was unfounded. Schoenheimer and Rittenberg had demonstrated the extensiveincorporation of deuterium from D2O into cholesterol in the mouse and concluded thatcholesterol must be synthesized by the joining of a number of small molecules.

The pathway for the biosynthesis of cholesterol from acetate, involving more than 30separate enzyme-catalyzed reactions, can now be found in every textbook of biochemistry. Adetailed review is beyond the scope of this essay. Here we will consider only the principallandmarks in its three major stages: 1) acetate to “activated isoprene”; 2) “activated isoprene”to squalene; and 3) squalene to cholesterol.

Bloch’s studies began with investigations of the overall process of formation of cholesterol inthe intact organism. Stimulated by a report from the German workers Sonderhoff and Thomas(26), indicating that acetate is efficiently converted into the sterols of yeast, Bloch began aseries of studies demonstrating the incorporation of specifically labeled acetate into cholesterolin the intact animal. These studies were continued and expanded after his move in 1946 to theDepartment of Biochemistry at the University of Chicago, where his good friend Earl Evans,also a product of Hans Clarke’s department, had become chairman.

I was a graduate student in the Department at this time, and so I came to know KonradBloch, first as a teacher and later as a colleague and friend. He was a man of personal qualitiescommensurate with his great abilities. His manner with students was friendly and easy. Hewas painstakingly generous in acknowledging the research contributions of his colleagues andof other laboratories. He was widely cultured, devoted to music, literature, and art.

In the mid-1940s, Bloch (23) was completely convinced of the truth of Lipmann’s dictum thatenergy-requiring biosynthetic reactions are driven by ATP, directly or indirectly. Before thisperiod the synthesis of peptide bonds had been observed only by reversal of the reactionscatalyzed by proteases. In a project quite unrelated to the cholesterol problem, he and hisstudents began to investigate the synthesis of the tripeptide glutathione as a possible modelof protein synthesis. They were indeed able to show that the assembly of glutathione requiresthe successive activation of glutamate and glutamylcysteine by ATP, but unfortunately themechanism proved to shed little light on the ribosomal synthesis of proteins.

Bloch was also very much aware of the potential power of microbial genetics for the analysisof metabolic pathways, and he enrolled as a student in the famous course in microbiologytaught by C. B. Van Niel at the Hopkins Marine Station in Pacific Grove, CA. When a mutantof the mold Neurospora crassa was isolated in Tatum’s laboratory that grew only when acetatewas added to the medium, Bloch was eager to follow this lead. He and his collaborators foundthat isotopically labeled acetate was converted to ergosterol in this mutant essentially withoutdilution of the isotope. Clearly the sterol could be built up entirely from acetate.

In the conversion of acetate to cholesterol, which of the carbon atoms of cholesterol werederived from the carboxyl group and which from the methyl group? Studies carried out over anumber of years in the laboratories of Cornforth and of Popjak, as well as of Bloch, achievedthe ambitious goal of defining the origin of each of the 27 carbon atoms of cholesterol as eitherthe methyl or the carboxyl carbon of acetate. This work placed important constraints onpossible structures of intermediates in the scheme.

It had been known for some time that squalene (a branched, acyclic hydrocarbon found inabundance in the livers of sharks) when fed to animals increases the levels of cholesterol intheir tissues. To test the idea that squalene might be a precursor of cholesterol, Bloch went tothe Biological Research Station in Bermuda to attempt the preparation of isotopically labeledsqualene in shark liver, but the shark proved to be an intractable subject for study (23). “AllI was able to learn was that sharks of manageable length are very difficult to catch and theiroily livers impossible to slice.” Back at the University of Chicago, however, his student RobertLangdon was able to prepare labeled squalene by feeding rats labeled acetate along withunlabeled squalene as an isotopic trap. Labeled squalene so obtained was then fed to rats andfound to be converted to cholesterol (27). This was an important result. In the dissection ofevery biosynthetic pathway, it is particularly helpful to identify an intermediate in the middleof the chain of reactions; the researcher can then trace the pathway both backwards andforwards. At this stage in his work, in 1954 Bloch moved to the Department of Chemistry atHarvard, where he was to remain for the rest of his career.

Squalene, containing 30 carbon atoms, could plausibly be considered to be built up from 6units of isoprene, a branched, unsaturated compound containing five carbon atoms. Isoprene


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was already known to be a building block of other naturally occurring hydrocarbons such asrubber, although the nature of the biologically active “isoprene donor” remained unknown.

Robinson (28) had suggested that squalene might be folded to form the basic structure ofcholesterol directly. Bloch, however, after illuminating discussions with his Harvard colleagueRobert Woodward considered that lanosterol, with a structure closely similar to cholesterol butwith three “extra” methyl groups, was likely to be an intermediate in this transformation.

Up to this point, Bloch’s experimental approach to the cholesterol problem had been largelyconfined to isotopic tracer studies with intact animals or with tissue slices in which cellularstructure was preserved intact, but now he turned increasingly to the study of cell-free enzymesystems. Rat liver homogenates, prepared by the methods developed by Nancy Bucher, werefound to catalyze the transformation of labeled squalene to lanosterol and of lanosterol tocholesterol. Although much work remained to be done, Bloch had established the landmarksfor the final stages of the biosynthesis of cholesterol (29).

The focus now was turned to the first stages of the pathway, the conversion of acetate to the“active isoprene donor.” A mutant strain of Lactobacillus acidophilus had been found to growonly when acetate was added to the medium. A substance that very efficiently replaced theacetate requirement was identified by workers at Merck, Sharpe and Dohme (30) as mevalonicacid (isolated as the lactone). Mevalonic acid was then shown to be a very efficient precursorof squalene and of cholesterol in homogenates of liver (31). These findings opened the way forthe elucidation of the reactions leading to the formation of the “active isoprene unit” of whichmevalonate was clearly the precursor. Progress in this area now became fast and furious withimportant contributions from the laboratories of Rudney, Lynen, Cornforth, and Popjak amongothers.

Bloch and his collaborators showed that the overall conversion of labeled mevalonic acid tosqualene in extracts of bakers’ yeast required ATP as well as reduced pyridine nucleotide andmanganese ions. His colleague Chen then discovered the phosphorylation of mevalonate to amonophosphate. The further conversion of this monophosphate to the important intermediatesisopentenylpyrophosphate and dimethylallylpyrophosphate was elucidated largely by work inLynen’s laboratory.

The synthesis of squalene via geranyl pyrophosphate and farnesyl pyrophosphate was nextdocumented. As shown by the early studies of Bloch, squalene is converted in a series of stepsto lanosterol, which after several further transformations gives rise to cholesterol.

It is impossible, of course, in this highly condensed account to do justice to the vast amountof work, still ongoing in laboratories over the world, that has led to our present knowledge ofthe biosynthesis of cholesterol. It was Bloch, however, who was a prime mover in all threephases of the problem. For this work he was awarded a Nobel Prize, with Feodor Lynen, in1964.

Working out the pathway for the assembly of the complex structure of cholesterol was anexemplary achievement of the era of biosynthesis, important not only because of the intrinsicinterest of its enzymology but also because of its significance for medicine. High levels of bloodcholesterol, characteristic of populations in developed countries, strongly increase the dangerof heart disease and stroke. An understanding of the detailed route of biosynthesis made itpossible to determine that the synthesis of mevalonate from HMG-CoA is a rate-making stepin the production of cholesterol. This advance made possible the development of drugs, thefamily of statins, that reduce levels of blood cholesterol with a minimum of toxic side effects.These drugs are among the most useful in modern medicine.

Konrad Bloch made outstanding contributions to fields other than the biosynthesis ofcholesterol, including the enzymic synthesis of fatty acids and the mechanism of enzyme action(23). He died on October 15, 2000 at the age of eighty-eight.

The development of any field of science is inevitably the work of many hands. Obviously,Lipmann, Schoenheimer, and Bloch cannot be regarded as single handedly transformingAmerican biochemistry. Their work was nonetheless a great gift to their adopted country anda shining manifestation of the international character of science.

REFERENCES1. Medawar, J., and Pyke, D. (2001) Hitler’s Gift, Arcade Publishing, New York2. Lipmann, F. (1953) Annu. Rev. Biochem. 54, 1–323. Lipmann, F. (1971) Wanderings of a Biochemist, Wiley-Interscience, New York4. Warburg, O. (1949) Wasserstoffuebertragende Fermente, Editio Cantor, Freiburg, Germany


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5. Lipmann, F. (1939) Nature 144, 33–346. Lipmann, F. (1941) Adv. Enzymol. 1, 99–1627. Lipmann, F. (1945) J. Biol. Chem. 160, 173–1908. Lipmann, F., and Kaplan, N. O. (1946) J. Biol. Chem. 162, 743–7449. Lipmann, F., Kaplan, N. O., Novelli, G., Tuttle, L. G., and Guirard, B. M. (1947) J. Biol. Chem. 167, 869–870

10. Lynen, F., and Reichert, E. (1951) Angew. Chem. 63, 47–4811. Hevesy, G. (1923) Biochem. J. 17, 439–44512. Kohler, R. E., Jr. (1977) Hist. Studies Phys. Sci. 8, 257–29813. Young, V. R., and Ajami, A. (1999) Proc. Nutr. Soc. 58, 15–3214. Hevesy, G. (1948) Cold Spring Harbor Symp. Quant. Biol. 13, 129–15015. Clarke, H. T. (1958) Annu. Rev. Biochem. 27, 1–1416. Palmer, W. (1966) in Nobel Lectures Chemistry 1922–1941, pp.333–338, Elsevier Science Publishing Co., Inc., New

York17. Lewis, G. N. (1934) Science 79, 151–15318. Schoenheimer, R., and Rittenberg, D. (1935) Science 82, 156–15719. Schoenheimer, R. (1949) The Dynamic State of Body Constituents, Harvard University Press, Cambridge, MA20. Bernhard, K., and Schoenheimer, R. (1940) J. Biol. Chem. 133, 707–71221. Rittenberg, D., and Schoenheimer, R. (1937) J. Biol. Chem. 121, 235–25322. Schoenheimer, R., Ratner, S., and Rittenberg, D. (1939) J. Biol. Chem. 127, 333–34423. Bloch, K. (1987) Annu. Rev. Biochem. 56, 1–1924. Tchen, T. T., and Bloch, K. (1956) J. Am. Chem. Soc. 78, 1516–151725. Windaus, H. O. (1996) in Nobel Lectures Chemistry 1922–1941, pp. 105–121, Elsevier Science Publishing Co., Inc.,

New York26. Sonderhoff, R., and Thomas, H. (1937) Ann. Chem. 530, 195–21327. Langdon, R. G., and Bloch, K. (1952) J. Biol. Chem. 200, 129–14428. Robinson, R. J. (1934) J. Chem. Soc. Ind. 53, 1062–106329. Bloch, K. (1965) Science 150, 19–2830. Wolf, D. E., Hoffman, C. H., Aldrich, P. E., Skeggs, H. R., Wright, L. D., and Folkers, K. (1956) J. Am. Chem. Soc.

78, 449931. Tavormina, P. A., Gibbs, M. H., and Huff, J. W. (1956) J. Am. Chem. Soc. 78, 4498–4499


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The Biotin Connection: Severo Ochoa, Harland Wood,and Feodor Lynen

Published, JBC Papers in Press, May 27, 2004, DOI 10.1074/jbc.X400005200M. Daniel LaneFrom the Department of Biological Chemistry, The Johns Hopkins University School of Medicine,Baltimore, Maryland 21205

Unique circumstances sometimes bring us into contact with individuals who will profoundlyinfluence us, particularly in our formative years. In this article I would like to reflect on thecircumstances that brought me into contact with three great biochemists, Severo Ochoa (1),Harland Wood (2), and Feodor Lynen (3). Each entered the field by a different route: Ochoa asa physician with an interest in physiology, Wood as a bacteriologist trained at Iowa StateUniversity, and Lynen as an organic chemist trained in the German tradition with Nobel Prizewinner, Heinrich Wieland.

They entered the field of biochemistry in the late 1930s when the race was on to discover newenzymes, cofactors, and metabolic cycles. Hans Krebs had formulated the tricarboxylic acidcycle in 1937 and ornithine cycle (now known as the urea cycle) in 1932, some B vitamins hadbeen found to function as cofactors or prosthetic groups of enzymes, and Rudolf Schoenheimer(Columbia University College of Physicians and Surgeons) had demonstrated the dynamicstate of tissue proteins using heavy isotopes of hydrogen and carbon (mid-1930s). This waswhere the action was and it attracted many of the brightest young minds into the field. Thiswas the arena in which Ochoa, Wood, and Lynen were early participants. Excited by discovery,they transmitted this excitement to their younger colleagues.

I was fortunate to have scientific associations and enduring friendships with each of them.My connection developed through the B vitamin, biotin, and its role in the reactions catalyzedby a family of biotin-dependent enzymes, notably carboxylases. The B vitamin, biotin, has aninteresting history not familiar to most scientists who now make use of it. Today, this vitaminis widely used along with avidin (or its cousin, strepavidin), the specific biotin-binding proteinfrom egg white, to probe biochemical phenomena. Biotinylation of proteins and nucleotides andthe use of avidin to “fish out” or detect these molecules from/in complex mixtures has foundgreat utility.

It is a curiosity that nature has brought together within the hen’s egg the richest source ofbiotin in the yolk and in the white, a “toxic” factor, avidin, which when fed to animals causesbiotin deficiency. In 1936, Kogl and Tonnis isolated 1.1 mg of biotin from more than 500pounds of egg yolk. Paul Gyorgy recognized that the distribution, fractionation behavior, andchemical properties of Kogl’s yeast growth factor and the anti-egg white injury factor in eggyolk (then called vitamin H) were similar. When Kogl’s pure biotin methyl ester becameavailable it was found to be extremely potent in protecting rats against “egg white (i.e. avidin)injury.” Within a few years Vincent Du Vigneaud and colleagues determined the structure ofbiotin, which cleared the way for an attack on the role of biotin at the molecular level.

By 1950 biotin had been implicated in a number of seemingly unrelated enzymatic processesincluding the decarboxylation of oxaloacetate and succinate; the “Wood-Werkman reaction”(discovered by Harland Wood (2)), i.e. the carboxylation of pyruvate; the biosynthesis ofaspartate; and the biosynthesis of unsaturated fatty acids. Of course, we now know that biotin

THE JOURNAL OF BIOLOGICAL CHEMISTRY Vol. 279, No. 38, Issue of September 17, pp. 39187–39194, 2004© 2004 by The American Society for Biochemistry and Molecular Biology, Inc. Printed in U.S.A.

ReflectionsA PAPER IN A SERIES COMMISSIONED TO CELEBRATE THE CENTENARY OF THE JBC IN 2005




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functions in each of these processes as a mobile “CO2 carrier” bound covalently to a carbox-ylase. The long sought after link between biotin and enzymatic function was provided byHenry Lardy at the University of Wisconsin. Lardy showed that liver mitochondrial extractscatalyzed the ATP- and divalent cation-dependent carboxylation of propionate (subsequentlyshown to be propionyl-CoA) to form succinate (4). Later work in the laboratory of Severo Ochoafound that the initial carboxylation product was methylmalonyl-CoA, an intermediate en routeto succinyl-CoA. The connection to biotin was made by Lardy with the finding that thepropionate-carboxylating activity was lacking in liver mitochondria from rats made biotin-deficient by being fed egg white, which of course contained avidin (KD(biotin) �10�15). Moreover,the failure of mitochondrial extract to catalyze the carboxylation of propionate was quicklycured by injecting the rats with biotin.

Upon joining the faculty at Virginia Polytechnic Institute in Blacksburg, Virginia in 1956,I decided to try to determine how propionate is metabolized in the liver. Because of its uniquefeatures, I settled on bovine liver as the tissue source of the enzyme system to address thisquestion, propionate being a major hepatic carbon source in ruminants. Unlike carbohydratedigestion by monogastric animals, ruminants digest carbohydrates in the rumen, the largeanaerobic fore compartment of their multi-compartmented “stomach.” Virtually all carbohy-drate is fermented in the rumen to short chain fatty acids, primarily acetate and propionate.Thus glucose, the major digestion product of carbohydrates in monogastric animals, is notavailable for absorption in ruminants. Propionate, produced in abundance by fermentation inthe rumen, is absorbed directly into the portal system and transported to the liver where it isthe major carbon source for gluconeogenesis, the pathway leading to glucose production.

My entry into this area coincided with Lardy’s report that propionate was somehow carbox-ylated to form succinate. I recall writing to Henry Lardy, and he referred me to Severo Ochoaat New York University School of Medicine. He knew that Ochoa was working on propionatemetabolism and had found that propionyl-CoA first became carboxylated to form methylma-lonyl-CoA and then was converted to succinyl-CoA. With some trepidation about competingwith the Ochoa laboratory, I decided to forge ahead and purify propionyl-CoA carboxylase frombovine liver mitochondria. For the reasons mentioned above bovine liver turned out to be anexcellent source of the enzyme. At that point I wrote to Severo Ochoa, and he generously gaveme a status report on their progress and put me in contact with the people in his laboratory(Alisa Tietz, Martin Flavin, and later, Yoshito Kaziro) who were working on the enzyme. Thisinitiated what was to be a long relationship with Severo Ochoa and also his colleague, YoshitoKaziro (now in Tokyo).

About that time I applied to the National Science Foundation for a research grant to supportmy work on propionate metabolism. The grant proposal was rejected because the reviewers feltthat I was really “in over my head” competing with the Ochoa laboratory and also because ithad been rumored that his laboratory had already crystallized the enzyme from muscle. I knewthat this was not true because in my correspondence with Ochoa he had indicated that thecrystals turned out to be pyruvate kinase, not propionyl-CoA carboxylase. After much anguishI wrote to the Head of the National Science Foundation Review Committee, Louis Levin,indicating that the Committee was mistaken: “the carboxylase had not been crystallized” andthat I thought it was inappropriate for the National Science Foundation to take a position ona grant application based on the size of the laboratory, rather than the merit of the proposal.A few weeks later I received a letter from Lou Levin indicating that the Study Section hadreversed its decision and that the grant would be funded. I doubt seriously if that could happentoday. Thus began my independent career in research and a developing relationship withSevero Ochoa.

In 1959, a paper by Lynen and Knappe appeared in Angewandte Chemie (5) (later publishedin full in Biochemische Zeitschrift (6)) that created tremendous excitement in my laboratory.The paper described the rather remarkable finding that �-methylcrotonyl-CoA carboxylase, abiotin-dependent carboxylase (involved in leucine catabolism in certain bacteria), catalyzedthe ATP-dependent carboxylation of “free” biotin in the absence of its acyl-CoA substrate. Theproduct was shown to be a labile carboxylated biotin derivative, later identified as 1�-N-carboxybiotin. Because biotin was believed to be a prosthetic group covalently bound to theenzyme and because free biotin exhibited an extremely high Km, Lynen proposed that the freebiotin had accessed the active site of the carboxylase and by mimicking the biotinyl prostheticgroup had gotten carboxylated.

Reflections: The Biotin Connection

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Shortly thereafter Don Halenz and I succeeded in purifying a related enzyme, propionyl-CoAcarboxylase, from bovine liver mitochondria. After convincing ourselves that it too was abiotin-dependent enzyme, we turned our attention to how the biotinyl group was attached tothe carboxylase and what enzymatic reactions were involved in its becoming attached to thecarboxylase. Dave Kosow, also in my laboratory at Virginia Tech, had just found that extractsof liver from biotin-deficient rats contained catalytically inactive propionyl-CoA apocarboxy-lase. Moreover, he demonstrated that a soluble ATP-dependent enzyme system in theseextracts from the livers of the biotin-deficient animals catalyzed the covalent attachment of[14C]biotin to the apoenzyme, thereby restoring its ability to carboxylate propionyl-CoA (7).

Moreover, Dave Kosow showed (7) that upon treating the 14C-biotinylated carboxylase withStreptomyces griseus protease, biocytin (i.e. �-N-biotinyl-L-lysine) was released. This meant, ofcourse, that the biotin prosthetic group had been linked to propionyl-CoA carboxylase throughan amide linkage to a lysyl �-amino group. A few years later it became evident that this long(�14 Å) side arm facilitates oscillation of the 1�-N-carboxybiotinyl prosthetic group betweencatalytic centers on the enzyme (7).

After completing those experiments I invited Severo Ochoa to visit Virginia PolytechnicInstitute and to present two lectures, which he graciously agreed to do. One of these talks dealtwith propionyl-CoA carboxylase and the other with the genetic code, the two major projectsunder way in the laboratory of Ochoa at the time. While he was in Blacksburg Dave and Ishowed him our results on the site of attachment of biotin to the enzyme. We gave him someof the protease and within a month of his return to New York City he confirmed our findingswith the heart propionyl-CoA carboxylase.

It was at this point in 1962 that I decided to take a sabbatical leave in Munich with FeodorLynen (known to his colleagues as “Fitzi”) at the Max-Planck Institut Fur Zellchemie where Icould continue the work on the enzymatic mechanism by which biotin became attached topropionyl-CoA carboxylase. Before leaving for Munich Dave Kosow and I developed anothermore potent apoenzyme system with which to investigate the “biotin loading” reaction. This

FIG. 1. Harland Wood, circa 1991. (Reprinted with permission of the Cleveland Plain Dealer newspaper.)


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system made use of Propionibacterium shermanii that expressed huge amounts of methylma-lonyl-CoA:pyruvate transcarboxylase, another biotin-dependent enzyme studied extensivelyby Harland Wood. Moreover, this organism had an absolute requirement for biotin in thegrowth medium, which when grown at very low levels of biotin produced large amounts of theapotranscarboxylase. The choice of the P. shermanii system turned out to be a good one. It sohappened that my stay in Munich coincided with Harland Wood’s sabbatical leave in Lynen’sInstitute. This was a two-fold bonus for me, first because Harland was the world’s expert onthis enzyme and second because it began a lasting personal relationship with him. He has beena role model for me ever since that period in Munich.

Harland (1907–1991) grew up on a farm near Mankato, Minnesota. He entered MacalesterCollege in Minnesota where he majored in chemistry and worked his way through college.While a student at Macalester, he met Milly Davis and in their third year of college theymarried (in 1929, the year of the stock market crash and beginning of the great depression ofthe 1930s). In those days this required a meeting (for approval I presume) with the Presidentof the college, who needn’t have been concerned as she was at his side for the next 62 years.They were an amazing couple, a cooperative inseparable team. My wife and I shared theirfriendship for more than 30 years. In 1931, Harland became a graduate student in bacteriologyin the laboratory of C. H. Werkman at Iowa State University in Ames, Iowa, where he madea discovery that was so controversial, although correct, that it was questioned by his thesisadviser Werkman as well as by leaders in the field of microbial metabolism including C. B. vanNiel. Harland had discovered (2) that heterotrophic organisms, such as the Propionibacteria,were able to fix CO2. Prior to this it was believed that only auxotrophs, i.e. chemosynthetic orphotosynthetic auxotrophs, could carry out the net synthesis of organic compounds from CO2.His discovery truly opened the area of enzymatic carboxylation in higher organisms. Aftercompleting his Ph.D. degree Harland (Fig. 1) did postdoctoral work at the University ofWisconsin with W. H. Peterson and then returned to Iowa State as a faculty member. Harlandwas an innovator and an improviser. While at Iowa State he decided to conduct CO2 fixationexperiments using 13CO2, but because of World War II restrictions he could not gain access toa mass spectrometer nor could he obtain “heavy” 13CO2. In true Woodsian style, he built hisown mass spectrometer and constructed a thermal diffusion column in the Science building atIowa State College (2). In 1946, Harland became Professor and Director of the BiochemistryDepartment at Western Reserve (now Case-Western Reserve) University. He ran the mostdemocratic department on record in which faculty salaries were determined by the faculty ata meeting where members voted on one another’s salary for the upcoming year!

Upon arriving in Munich in August of 1962, I indicated to Lynen that I would like toinvestigate the P. shermanii “biotin loading” enzyme system, and he agreed with my proposal.Because Harland Wood was already at the Institute, I got his advice on growth conditions andfor large scale preparations of the transcarboxylase (actually, the apotranscarboxylase). BothLynen and Wood were quite enthusiastic about the project. It turned out that by growingP. shermanii in biotin-deficient medium the bacteria produced as much of the apotranscar-boxylase as the holotranscarboxylase when the organism was grown on normal/biotin-contain-ing medium. Within a short time I was able to resolve and purify both the apotranscarboxylaseand the synthetase that catalyzed loading biotin onto the apoenzyme (7). Dave Young, apostdoctoral fellow who had recently completed his medical training at Duke University, andKarl Rominger, a Ph.D. candidate under Lynen’s direction, collaborated with me on thesestudies. Finally, we proved that the synthetase catalyzed a two-step reaction in which the firststep involved the ATP-dependent formation of biotinyl-5�-AMP and pyrophosphate after whichthe biotinyl group was transferred from the AMP derivative to the appropriate lysyl �-aminogroup of the apotranscarboxylase.

While in the midst of these studies, a controversy developed regarding the site at whichbiotin became carboxylated during catalysis. It was suggested that HCO3

� became incorpo-rated into the 2�-position of the ureido ring of the covalently bound biotinyl prosthetic groupof biotin-dependent enzymes and that the 2�-carbon was then transferred to the acceptorsubstrate. It was suggested that Lynen’s experiments (referred to above) had been done withfree biotin and not the biotinyl prosthetic group covalently linked to the carboxylase. Such amechanism would have necessitated opening and then closing the ureido ring of biotin duringthe course of the reaction, which to a chemist like Lynen didn’t make chemical sense.Moreover, this proposal was inconsistent with the known lability of free 1-N-[14C]carboxy-


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biotin. We knew from my earlier studies that enzyme-14CO2�, presumably enzyme-biotin-

14CO2� (prepared by incubating propionyl-CoA carboxylase with H14CO3

� and ATP-Mg2�), waseven less stable than free 1-N-carboxy-[14CO2

�]biotin. so we set out to address the issue headon using propionyl-CoA carboxylase as the source of enzyme-biotin-14CO2

�. The previous springbefore going to Munich, I had found that enzyme-14CO2

� (derived from propionyl-CoA carbox-ylase) could be stabilized by methylation with diazomethane, i.e. enzyme-14CO2

� was labile toacid before but was stable after methylation. Moreover, digestion of methylated enzyme-14CO2

�

(enzyme-14CO2-CH3) with S. griseus protease produced a single radioactive derivative, pre-sumably methoxy-[14C]carbonyl-�-N-biotinyl lysine. This product had chromatographic prop-erties similar, but not identical, to �-N-biotinyl lysine. Because I did not have the authenticcompound for comparison, these experiments could not be completed at the time. Fortunately,Joachim Knappe, a former member of Lynen’s research group now at the University ofHeidelberg, had synthesized the derivative and provided Lynen with a sample. Thus, we wereable to verify the presumptive identification. This proved that the covalently bound biotinylprosthetic group, like free biotin, was carboxylated at the 1�-N position (8). Shortly thereafter,Knappe in Heidelberg and Harland Wood on sabbatical in Lynen’s laboratory in Munichshowed using a similar approach that the carboxybiotin prosthetic groups of �-methylcrotonyl-CoA and transcarboxylase, respectively, had identical structures (9). Taken together thesestudies proved unequivocally that the site of carboxylation of biotin was on the 1�-N of thebiotinyl prosthetic group.

By this point in my sabbatical in Lynen’s Institute, I began to recognize certain habits of “theChief.” For example, he had the habit of working in his office until late in the afternoon. Then,around dusk, i.e. 6:00–6:30 p.m., he would emerge to make “rounds” in the Institute, movingfrom one bench to the next to survey the day’s progress or lack of it. Of course not one of the�30 investigators would consider leaving until after he had passed through. He ran a “tight

FIG. 2. Feodor (“Fitzi”) Lynen, circa 1980. (Reprinted with permission of the Max-Planck Gesellschaft.)


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ship”! Fitzi had an uncanny memory and could recall details of experiments done weeksearlier.

Lynen (1911–1974) (3) (Fig. 2) was born and spent his entire life in Munich and environs. Hereceived his doctoral training in organic chemistry at the University of Munich with HeinrichWieland (Nobel Prize in Chemistry in 1927), graduating in 1937. He then married Wieland’sdaughter, Eva. He was spared the ravages of World War II because of a serious skiing accident,which left him with a persistent limp. Perhaps Lynen’s most important contribution was thediscovery of acetyl-CoA, the elusive molecule “active acetate,” sought after by many investi-gators including Fritz Lipmann, David Nachmansohn, and Severo Ochoa. Ochoa had discov-ered “condensing enzyme,” now known as citrate synthase, which catalyzed the formation ofcitrate from “active acetate” and oxaloacetate. These discoveries led to an important collabo-ration between Lynen and Ochoa in which they proved that citrate synthase used acetyl-CoA,along with oxaloacetate, to form citrate. These findings finally answered the question of how“active acetate” entered the citric acid cycle. In 1964 Lynen received the Nobel Prize (withKonrad Bloch) in Physiology or Medicine for his work on “the mechanism and regulation ofcholesterol and fatty acid metabolism.”

Lynen had strong connections to the United States. Many Americans came to his Instituteto do postdoctoral work or sabbaticals. During the period that Harland Wood and I spent inMunich, the other Americans in the group included Esmond Snell, on sabbatical leave fromBerkeley, David Young, Walter Bortz, Dick Himes, Paul Kindel, Martin Stiles, and EdWawskiewicz. Although Fitzi Lynen was a hard driving biochemist, he did like to socialize overa beer or a martini. On Friday afternoons Harland would often bring a half-gallon bottle ofGilbey’s gin to the Institute and prepare martinis in the second floor laboratory.

Shortly after returning from Munich in the Summer of 1963, I received a phone call fromSevero Ochoa, who asked if I might be interested in joining the faculty of his department atNew York University School of Medicine in New York City. My wife, Pat, and I had someconcern about moving from the bucolic setting of Blacksburg, Virginia (where we could see 20miles from our living room window) to the big city. Nevertheless, we relished the newchallenges ahead and were ready for a change in lifestyle. We loved New York City and neverregretted having made the decision. Severo Ochoa helped make it worthwhile.

Severo Ochoa (1905–1993) (1, 10) (Fig. 3) was born in Luarca, Spain, the youngest of sevenchildren. His father was a lawyer and businessman. He completed his M.D. degree (withhonors) at the University of Madrid. Though never having studied with him, he was inspiredby Ramon y Cajal, the Spanish neuroanatomist and Nobel Prize winner (1906). Following

FIG. 3. Severo Ochoa with colleagues viewing an enlargement of an electron micrograph of acetyl-CoA car-boxylase (1966). From right to left: Albrecht Kleinschmidt, Erwin Stoll, Severo Ochoa, and Dan Lane.


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medical school (1929–1931) Ochoa joined Otto Meyerhof’s laboratory in Heidelberg where heworked on muscle glycolysis. His early days in science were marked by the upheavals inEurope leading up to World War II. At the time of the Spanish civil war in 1936, he left Spainfor Heidelberg for the second and final time. Then in 1938, because of the turmoil in Germany,he moved to Oxford University in England to work in Professor Rudolph Peter’s unit. In 1941he came to the United States where he joined Carl and Gerty Cori at Washington Universityin St. Louis.

In his comments at the Nobel Prize banquet in 1964, Ochoa spoke of those who hadinfluenced him most.

I was deeply influenced by my great predecessor Santiago Ramon y Cajal. I entered Medical School too lateto receive his teachings directly but, through his writings and his example he did much to arouse myenthusiasm for biology and crystallize my vocation. Among the great names that adorn the roll of Nobelprize-winners in Medicine is that of Otto Meyerhof, my admired teacher and friend, to whose inspiration,guidance and encouragement I owe so very much. I was very fortunate to have worked also under theguidance of other great scientists and I wish to acknowledge my indebtedness to Sir Rudolph Peters andto Nobel prize winners Carl and Gerty Cori who did so much to add new dimensions to my scientificoutlook and enlarge my intellectual experience.

The seven years (1964–1970) I spent in Ochoa’s department were among the most excitingof my scientific career. It was a small department with only a handful of faculty, which at thattime included Charles Weissman, Bob Warner, Bob Chambers, Albrecht Kleinschmidt, andSevero. Upon arriving at New York University Medical School in August of 1962, Severo askedme to give 15 lectures in the first year medical student biochemistry course the next month.This course was Ochoa’s pride and joy and he and the faculty attended every lecture. (Inretrospect, I feel that this is an excellent way to ensure quality control in teaching.) At thetime, however, I hadn’t relished the idea of having a Nobel prize winner (1964, with ArthurKornberg, Ochoa’s first postdoctoral fellow) in the audience for the first 15 lectures in my newscientific home. Despite knowing that my first few lectures at New York University were notparticularly good, after the lecture Severo put his hand on my shoulder and said, “That was anexcellent lecture, Dan.” I knew that it hadn’t been, but I did appreciate the encouragement.This was typical of Severo’s behavior toward young scientists in whom he had confidence. Isuspect that his response reflected the encouragement he had received from his mentorsduring his development.

Every afternoon at 3:00 p.m. we took a break for coffee in the department library where wediscussed the latest results of our experiments or a hot new paper. Because the faculty wassmall, these were informal gatherings, which created a sense of camaraderie. Severo neverfailed to show up for these sessions. We could always count on Charles Weissman for a good,often slightly “off color” joke. “Have you heard the one about the ——?” Because of his innateability at story telling, Charles was a favorite lecturer of the medical students. His timing wasimpeccable.

Severo had a princely presence in part because of his carriage, tall stature, and silver hair.At national/international meetings, when he walked into a room he attracted hushed atten-tion. Despite this, he had a warm personality and showed genuine concern for his colleagues,associates, and students.

It is natural that we feel a closeness to those to whom we are related through researchinterests. In Hans Krebs book, Reminiscences and Reflections (11), he illustrates the scientificgenealogy leading to Ochoa.

We talk rather loosely these days about “impact factor” (and citation index) in evaluating theworth of one’s publications, but it is the excitement and joy of doing science, rather than therecognition itself, that motivates us.

Research today moves at great speed. Communication is rapid, publication is rapid, and oneis left with the impression that everything of importance was done in the past 10 years.However, science is built stepwise on the shoulders of those who came before us. Little istaught today as to how each of our particular areas of the biological sciences developed. Formany students the “important stuff” now goes back into the past for only 7–8 years. Mostonline scientific journals go back only 7–8 years. Fortunately, the Journal of BiologicalChemistry is the exception and is to be commended, because it is online all the way back to thepoint of its origin in 1905. These Reflections may be a sign of recognition that the history ofdiscovery still has importance.


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Acknowledgment—I thank my wife, Pat Lane, who assisted with this article and shared these friendships and experienceswith me.

Address correspondence to: [email protected].

REFERENCES1. Ochoa, S. (1980) Annu. Rev. Biochem. 49, 1–302. Wood, H. G. (1985) Annu. Rev. Biochem. 54, 1–413. Lynen, F. B. (1964) Information available on the Nobel Museum Web Site: www.nobel.se/4. Lardy, H. A., and Adler, J. (1956) J. Biol. Chem. 219, 933–9425. Lynen, F., Knappe, J., Lorch, E., Jutting, G., and Ringelmann, E. (1959) Angew. Chem. 71, 481–4866. Knappe, J., Ringelmann, E., and Lynen, F. (1961) Biochem. Z. 335, 168–1767. Moss, J., and Lane, M. D. (1971) Adv. Enzymol. Relat. Areas Mol. Biol. 35, 321–442 (a review article)8. Lane, M. D., and Lynen, F. (1963) Proc. Natl. Acad. Sci. U. S. A. 49, 379–3859. Lynen, F. (1967) Biochem. J. 102, 381–400

10. Kornberg, A., Horecker, B. L., Cornudella, L., and Oro, J. (eds) (1975) Reflections on Biochemistry, pp. 1–14,Pergamon Press, New York

11. Krebs, H. A. (1981) Reminiscences and Reflections, Oxford University Press, Oxford, UK


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