coupling of the biosynthesis of fatty acids and fatty alcohols

ARCHIVES OF BIOCWEMI~TRY AND BIOPHYSICS Vol. 186, No. 1, February, pp. 77-83, 1978

Coupling of the Biosynthesis of Fatty Acids and Fatty Alcohols

C. 0. ROCK,’ VERONICA FITZGERALD, AND FRED SNYDER

Medical and Health Sciences Division, Oak Ridge Associated Universities, Oak Ridge, Tennessee 37330

Received September 6, 1977; revised October 31, 1977

A cell-free system for the biosynthesis of fatty alcohols in the pink portion of the rabbit harderian gland is described. The radiolabeled substrates for the fatty acid reductase were generated using soluble fatty acid synthase from the gland in the presence of acetyl-CoA, malonyl-CoA, and NADPH. Harderian gland microsomes, ATP, and Mg2+ were absolute requirements for the synthesis of fatty alcohols and if any of these components were deleted from the assay mixture, no alcohols were detected. We were also unable to detect formation of fatty alcohols if acyl-CoAs were substituted for fatty acid synthase with either NADPH or NADH as reducing agents. The reductase was localized in the microsomal fraction and appears to be on the cytosol-membrane interface of the vesicles, as indicated in experiments using detergents and trypsin. The fatty alcohols formed by the system had the same chain length distribution as the fatty acids made by the fatty acid synthase. The alkyl moieties of the ether lipids in the harderian gland are exclusively saturated and the properties of the alcohol-synthesizing system described in this report can account for the observed exclusion of unsaturated alkyl moieties from the ether lipids of this gland.

Trace amounts of free fatty alcohols have been detected in a variety of mammalian tissues and neoplasms (l-3); however, they are most commonly found as components of wax esters in specialized glands (4-6) and in the skin (for a review, see Ref. 7). Also, fatty alcohols are utilized for the formation of the ether bond in glycerolipids by a microsomal enzyme (al- kyldihydroxyacetoneP synthase) that catalyzes the substitutio’n of a fatty alcohol for the acyl group of acyldihydroxyacetone phosphate (for a review, see Ref. 8).

Although the biosynthesis of fatty alcohols is of central importance in the formation of waxes and alkyl glycerolipids, rel- atively little is known about the enzymes responsible for their biosynthesis in mammalian cells. Snyder and Malone (9) re- ported that fatty acyl-CoAs are reduced to alcohols by microsomal preparations from mouse preputial gland tumors and that NADPH is required for the reduction. Fer- rell and Kessler (10) proposed a scheme

1 Present address: Department of Molecular Bio- physics and Biochemistry, Yale University, New Haven, Connecticut 06510.

for the interconversion of fatty acids and aldehydes in liver that involves both soluble and particulate enzymes. However, since liver does not contain appreciable quantities of waxes or ether lipids, the relationship of these activities to those responsible for supplying alcohols to these pathways is problematical. The most ex- tensively studied mammalian system has been bovine cardiac muscle, where two soluble enzymes are thought to work in concert in the production of fatty alcohols (11-13). The first enzyme reduces fatty acyl-CoA to the corresponding aldehyde and requires NADH as the reductant. A second soluble enzyme catalyzes the reduction of long-chain aldehyde to alcohols and is specific for NADPH. Other specific oxi- doreductases that utilize long-chain ali- phatic compounds have been studied in other mammalian tissues (14-19).

The pink portion of the rabbit harderian gland contains a preponderance of alkyl glycerolipids (20-22) and is actively en- gaged in the secretion of these lipids (23, 24). Accordingly, we have found this gland to be a rich source of the enzymes involved

77 0003-9861/78/1861-0077$02.00/O Copyright 0 1978 by Academic F’roee, Inc. All rights of reproduction in any form reserved.

78 ROCK, FITZGERALD, AND SNYDER

in the biosynthesis of ether-linked lipids (25-28). A characteristic feature of the alkyl moieties of these lipids is that they are exclusively saturated and are com- posed primarily of C,,:, and C,,:, alkyl chains (20-22). Studies on the substrate specificity of alkyldihydroxyacetone? synthase (29) have shown that this enzyme is capable of catalyzing the incorporation of a wide range of fatty alcohols into alkyldihydroxyacetone-P; a similar conclusion can be reached from in uiuo investigations (30, 31). These data suggest that the com- position of the alkyl moieties in ether lipids is controlled at the level of fatty alcohol synthesis. In this report, we describe a cell-free system from the harderian gland that is capable of synthesizing fatty alcohols. The properties of this system are similar, but not identical, to the alcohol-synthesizing system described by Khan and Kolattukudy (32) for Euglena grucilis and can account for the exclusion of unsaturated alkyl moieties from the ether lipids of the harderian gland.

EXPERIMENTAL PROCEDURES

Materials. [1-“C]Acetyl-CoA (sp act, 54 Ci/mol) was purchased from New England Nuclear Corp. Cholic acid, deoxycholic acid, Triton X-100, MgC&, and Tris were obtained from Sigma Chemical Co. ATP, CoA, malonyl-CoA, NADP, NADH, glucose B-phosphate, glucose 6-phosphate dehydrogenase, and dithiothreitol (D’lT12 were from P-L Biochemi- cals.

S&cellular fractionation. We excised the harderian glands from adult male New Zealand white rabbits obtained from Dutch Land Laboratory. The pink portions of the gland were separated from the white portion and then homogenized in a buffer containing 50 mM phosphate (pH 6.81, 50 mM KCl, 1 mM DTT, and 0.25 M sucrose. The resulting suspen- sion was centrifuged at 15,000g for 10 min and the pellet and fat layer were discarded. The remaining supernatant was then centrifuged at 105,OOOg for 90 min and used for the preparation of the fatty acid synthase; the pellet (microsomes) was resuspended in the homogenization buffer and centrifuged again at 105,OOOg for 90 min. The microsomes were then suspended in the homogenization buffer, divided into aliquots, and stored at -20°C. These microsomal preparations, used as the source of the acyl reductase, maintained their activity for at least 1 month under these storage conditions. The protein

z Abbreviation used: Dl”l’, dithiothreitol.

contents of all preparations were determined by the method of Lowry et al. (33).

Preparation and assay of the fatty acid synthase. The 105,OOOg supernatant was made 40% in (NH&SO, and then centrifuged at 10,OOOg for 10 min. The pellet contained greater than 95% of the total fatty acid synthase activity present in the 105,OOOg supernatant. The pellet was suspended in a buffer containing 50 mM phosphate (pH 6.8), 50 rnM KCl, and 1 mM D’lT and centrifuged at 105,OOOg for 1 h. This step was necessary to remove residual microsomal contamination from the sample. The supernatant was then applied to a column packed with Sephadex G-25, to remove residual (NH&SO,, and the void volume was collected. This preparation served as the source of fatty acid synthase in our experiments. The enzyme was maintained in a buffer of 0.1 M phosphate (pH 6.81, 50 mM KCl, and 10 mM DTT at 4°C.

Fatty acid synthase was assayed using a mixture containing phosphate (0.1 M, pH 6.81, KC1 (50 mM), DTT (1 mM), [lJ4Clacetyl-CoA (4 PM), malonyl- CoA (50 PM), NADP (1 mM), glucose 6-phosphate (5 mM), glucose 6-phosphate dehydrogenase (10 units), and fatty acid synthase (45 pg of protein) in a final volume of 1 ml. The reaction mixtures were incubated for 10 min at 37°C and then stopped by adding 1 ml of 2 N KOH. The incubation tubes were sealed and heated in a boiling water bath for 1 h. After acidification with 6 N HCl, the products were extracted twice with two 2-ml portions of ethyl ether. The ethyl ether was removed under nitro- gen and the products were dissolved in chloroform for radioactivity assay (34, 35). The reaction was linear from 10 to 90 pg of protein.

Assay of fatty alcohol synthesis. Incubations contained fatty acid synthase (45 pg of protein), microsomes (20 pg of protein), NADP (1 mr&, glucose 6- phosphate (5 mM), glucose 6-phosphate dehydrogenase (10 units), malonyl-CoA (50 PM), Il-14Clacetyl- CoA (4 /LM), DTT (1 mM), ATP (6 mr.r), Mg*+ (5 mM), and phosphate (0.1 M, pH 6.81 in a final volume of 1 ml. The reaction mixtures were incubated for 10 min at 37°C and terminated by adding 1 ml of 2 N KOH. Tubes were sealed and heated in a boiling water bath for 1 h. After acidification with 6 N HCl, lipids were extracted twice with two 2-ml portions of ethyl ether. Products were resolved by thin-layer chromatography on silica gel G layers developed in either hexane:ethyl ether:acetic acid (60:40:2, v/v/v) or hexane:ethyl ether:NH,OH (60:40:2, v/v/v). The distribution of “C products was determined by zonal profile scanning and area scraping procedures (34, 35).

Trypsin digestion of membranes. We incubated the microsomes in 50 mM Tris-HCl (pH 7.41, 50 mM KCl, and 0.25 M sucrose for 10 min at 37°C in the absence of any additions (control) and in the presence of either 50 pg of trypsin/mg of microsomal

COUPLING OF BIOSYNTHESIS OF FATTY ACIDS AND ALCOHOLS 79

protein, 0.05% deoxycholate, or both (36). Proteolysis was stopped by cooling on ice. The suspensions were centrifuged at 105,OOOg for 90 min; the pellets produced were resuspended in a buffer of 50 mM phosphate (pH 6.6), 1 rnr.r DTT, and 50 mM KC1 and assayed for activity in the fatty alcohol synthesis assay system described above. Cytochrome b, was measured as described by Omura and Sate (37).

RESULTS

Properties of fatty acid synthase in harderian gland. The properties of the fatty acid synthase in the harderian gland were found to be very similar to those of the fatty acid synthases from avian (38) and mammalian (39) livers. The enzyme ex- hibited a sharp pH optimum at pH 6.8 and required malonyl-CoA and NADPH, as well as acetyl-CoA (Table I). The enzyme was rapidly inactivated when stored in low ionic strength buffers, alkaline pH, in Tris, or in the absence of DTT. The enzyme was strongly inhibited by N-ethylmaleimide (Table I) and other sulfhydryl inhibitors. Extraction of the assay system by the procedure of Bligh and Dyer (401, rather than by saponifica- tion procedure described under Experi- mental Procedures, followed by chromatography on silica gel G layers developed in hexane:ethyl ether:acetic acid (60:40:2, v/v/v) demonstrated the products of the reaction to be free fatty acids. The addition of ATP and Mg*+ to the incubations did not reduce the enzymatic activity and did not alter the nature of the products.

Components of the fatty alcohol-synthesizing system. When ATP, Mg*+, and har-

TABLE I COFACTOR REQUIREMENTS FOB THE PARTIALLY

PURIFIED FATTY ACID SYNTHABE

System Nanomoles of fatty acid per

minute per milligram of

protein

Completea 3.5 -NADPH co.1 - Malonyl-CoA co.1 +ATP, MgZ+ 3.4 +iV-Ethylmaleimide (1 mr.r) co.1

D The complete system contained fatty acid synthase, the NADPH-generating system, [l-Wlacetyl- CoA, and malonyl-CoA in phosphate buffer (pH 6.8) as described under Experimental Procedures.

derian gland microsomes were added to the fatty acid synthase assay system, fatty alcohols were detected. ADP or AMP could not substitute for ATP in the reaction, nor did the addition of NADH to the system result in any increase in alcohol formation (Table II). The rate of synthesis of fatty alcohols was also dependent upon the presence of microsomes in the incubations (Table II), and Fig. 1 illustrates the dependence of fatty alcohol synthesis on microsomal protein concentration in the assay system. These data and the results described in the previous section indicate

TABLE II COFACTOR REQUIREMENTS FOR THE MICROSOMAL

REDUCTABE RESPONSIBLE POR FATTY ALCOHOL SYNTHESIS

System Picomoles of fatty alcohol formed per

10 min

Complete” 16.3 -ATP <l - M&+ <l -ATP, Mg2+ <l -ATP, +ADP <l -ATP, +AMP <l +NADH 18.5 -Fatty acid synthase <2 - Microsomes <I

L1 The complete system contained fatty acid synthase, harderian gland microsomes, [l-Wlacetyl- CoA, malonyl-CoA, NADPH, ATP, and MgZ+ as described under Experimental Procedures.

FIG. 1. Dependence of fatty alcohol synthesis on microsomal protein concentration. Incubations contained fatty acid synthase (45 pg), W*Clacetyl- CoA, malonyl-CoA, NADPH, ATP, and MgZ+ as described under Experimental Procedures.


that the microsomal cell fraction is the esters in hexane:ethyl ether:acetic acid source of the reductase. (80:20:1, v/v/v). Table III shows the chain

We have made repeated attempts to length distribution of the labeled fatty demonstrate the reduction of acyl-CoAs acids as determined by gas-liquid chro- by harderian gland microsomes without matography (41). Palmitic acid is the ma- success. Incubation of microsomes with [l- jor product, with smaller amounts of C,,:, 14C]palmitoyl-CoA (or [ l-14C]palmitic acid, and C,,:, chains. When harderian gland ATP, CoA, and Mg*+) and NADPH or microsomes were added to the assay, 14C- NADH did not result in the formation of labeled fattv alcohols were detected (Fia. fatty alcohols. The substitution of [l- “Clpalmitoyl-CoA for [l-14Clacetyl-CoA in the standard fatty alcohol-synthesizing system also did not produce fatty alcohols. Destruction of the acyl-CoAs by acyl-CoA hydrolase in the microsomal preparation is an unlikely possibility, since the methods we used to add the acyl-CoAs have been shown to be effective in assaying other acyl-CoA-utilizing enzymes in the harderian gland microsomes (25-27). Al- though the participation of acyl-CoAs in the reaction cannot be completely ruled out by these data, it would appear that exogenously added acyl-CoAs are not ac- cessible to the microsomal reductase.

Identification of ‘4c products. Fatty acids were the only radioactive products detected in the fatty alcohol-synthesizing system when harderian gland microsomes were deleted from the incubations (Fig. 2A). Methyl esters of these fatty acids cochromatographed with authentic methyl

4,

3-

F! 0 x 2- b

l-

2

I;

.I 0 1 2 3 4 5 6 7 6 9 10 11 12 13 14 15 16 17 I8

O~rtance From Ongm lcm)

2B). In addition to the fatty alcohols; an- other peak of radioactivity was observed that had chromatographic properties similar to those of a fatty aldehyde. However, only trace amounts of this component were detected and, therefore, it could not be analyzed further. Trimethylsilyl ethers of the fatty alcohols were prepared (42) and these derivatives cochromatographed with authentic trimethylsilyl ether derivatives of fatty alcohols on silica gel G layers developed in hexane:ethyl ether (90:10, v/ v). Gas-liquid chromatography revealed that the radioactivity present in the fatty alcohol fraction was distributed according to the chain length pattern found for the fatty acids (Table III); hexadecanol was the major component.

Effect of detergent and trypsin treatment on fatty alcohol synthesis. Since other enzymes in the ether lipid pathway in the harderian gland have been found to be markedly stimulated by detergents (27,

4

3

%

x2 z

1

2

1

B

1

A 3 00 J 0 1 2 3 4 5 6 7 8 9 10 1, 12 13 14 15 16 17 18

Oistamx From Origin IcmJ

FIG. 2. Zonal profile scans of the products of the harderian gland fatty alcohol-synthesizing system in the absence of microeomes (A) and in the presence of microsomes (B). Samples were chromatographed on silica gel G layers developed in hexane:diethyl ether:acetic acid (60:40:2, v/v/v). Radioactive peaks were identified as: fatty alcohol (Peak 11, fatty acid (Peak 2), and fatty aldehydes (Peak 3).


TABLE III

DISTRIBUTION OF RADIOACTIVITY AMONG CHAIN LENGTHS OF FATS ACIDS AND ALCOHOLS

SYNTHESIZED BY THE HARDERIAN GLAND SYSTEM

Chain length Percentage of total 14C

Fatty acids Fatty alcohols

14:o 1.0 Trace 16:0 86.5 85.4 l&O 12.5 14.6

28), we examined the effect of detergents on the fatty alcohol-synthesizing system. The results of these experiments are sum- marized in Table IV and, in contrast to other enzymes in the pathway (27, 281, the detergents inhibited fatty alcohol synthesis. Deoxycholate strongly inhibited both fatty alcohol synthesis and the fatty acid synthase (Table IV). Cholate and Tri- ton X-100 did not affect the fatty acid synthase, but markedly decreased the formation of fatty alcohols by the system. Microsomes were also incubated in the absence of any additions (control) or in the presence of trypsin, deoxycholate, or both as described under Experimental Pro- cedures. Treatment of the microsomes with trypsin alone produced a significant decrease in the acyl reductase activity recovered in the microsomes (Table V). In this regard, the acyl reductase behaved similarly to cytochrome b5, a marker protein for the cytosolic surface of microsomes (43). These data indicate that the protein responsible for the reduction of the fatty acids to fatty alcohols is located on the cytosolic surface of the microsomes.

DISCUSSION

In this report we describe a cell-free system for the biosynthesis of fatty alcohols in the harderian gland that can account for the observed distribution of alkyl chains in this gland. Unsaturated alcohols are not synthesized, since the alcohols are derived only from acyl groups formed from fatty acid synthase, not from acyl-CoAs or free fatty acids. Coupling of a microsomal fatty acid synthase to a microsomal acyl reductase has previously been noted in Euglena grucilis (32). Only 10-14s of the total fatty acid synthase in the harderian gland is associated with the microsomal

TABLE IV

EFFECT OF DETERGENW ON THE FATTY ALCOHOL- SYNTHESIZING SYSTEM”

Detergent added Picomoles Picomoles of fatty al- of total cohol per products

10 min per 10 min

None 16.2 140 Deoxycholate (2.0 mM) 0.8 18 Cholate (4.0 rnhb) 2.0 135 Triton X-100 (0.012%) 2.2 143

a The complete system contained fatty acid synthase, harderian gland microsomes, [l-Wlacetyl- CoA, malonyl-CoA, NADPH, ATP, and MgZ+ aa described under Experimental Procedures.

TABLE V

EFFECT OF TRYPSIN AND/OR DEOXYCHOLATE TREATMENT ON FATTY ALCOHOL SYNTHECW AND

CYTOCHROME b, IN HARDERIAN GLAND MICROSOMES

Assay system Percentage of control activity remaining in pellet after treatment

with

Deoxy- Trypsin Deoxy- cholate cholate

plua trypsin

Fatty alcohol syn- 81 4 4 thesis”

Cytochrome b, 100 9 6

a The complete system contained fatty acid synthase, harderian gland microsomes, [l-Wlacetyl- CoA, malonyl-CoA, NADPH, ATP, and Mg2+ aa described under Experimental Procedures.

cell fraction, which is not sufficient to account for the observed alcohol-synthesizing activity. ATP was also required in this system to bring about the coupling of fatty acid synthase to the reductase. The Mg2+ requirement was not investigated; however, this cation was present in all incubations (32). The E. grucilis system also differs from the one described in this report in that it required NADH for the formation of alcohols (321, whereas NADPH is the only reducing agent required in the harderian gland system. Furthermore, in the gland, the coupling occurs between the soluble synthase and the microsomal reductase, whereas in Eu- glena the coupling occurs between a tightly bound microsomal fatty acid synthase (different from the soluble synthase) and the microsomal reductase.


The role of ATP in the harderian gland cell-free system is not clear. One possible role may be the activation of fatty acids generated by the synthase to provide CoA derivatives to the microsomal reductase. However, we have been unable to demonstrate fatty alcohol synthesis in the harderian gland from acyl-CoAs or in incubations containing an acyl-CoA-generating system. Other activated compounds, such as acyl phosphates or acyl adenylates, are possible candidates for the intermediate in this reaction. Since ATP could also function as a coupling agent for bringing together the fatty acid synthase and the microsomal reductase, the substrate for the reductase could actually be the fatty acid bound to the acyl carrier protein portion of the synthase. We are continuing the investigation of this system to identify the intermediate(s) in the reaction.

ACKNOWLEDGMENTS

This work was supported by the United States Energy Research and Development Administration, the National Cancer Institute (Grant CA11949-08), the American Cancer Society (Grant BC-‘IOF), and a National Cancer Institute Training Grant (COR) (Grant CA05287-03).

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coupling of the biosynthesis of fatty acids and fatty alcohols

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