THE JOURNAL 01‘ B~WGICAL Cmmsmv Vol. 253, No. 5, Issue of March 10, pp. 1522-1528, 1978

Printed m U.S.A.

Rat Liver Peroxisomes Catalyze the /3 Oxidation of Fatty Acids*

(Received for publication, June 22, 1977)

PAUL B. LAZAROW

From The Rockefeller University, New York, New York 10021

Peroxisomes were purified by differential and equilibrium density centrifugation from the livers of rats treated with clofibrate to enhance their peroxisomal system of fatty acid oxidation. These purified peroxisomes were tested for the presence of crotonase, /3-hydroxybutyryl-CoA dehydrogen- ase and thiolase using spectroscopic techniques that utilize the characteristic absorption bands of the appropriate 4- carbon acyl-CoA substrates. All three enzymes were found. Analysis of the fractions from equilibrium density centrifu- gation revealed major peaks of these enzyme activities in peroxisomes and excluded contamination by mitochondria as an explanation of the results. In the presence of excess CoA the purified peroxisomes oxidized palmitoyl-CoA to acetyl-CoA, and reduced NAD, with a 1:5:5 stoichiometry. The peroxisomes were inactive with butyryl-CoA and less active with octanoyl-CoA than with lauroyl-CoA or palmit- oyl-CoA; they appear specialized for the /3 oxidation of long chain fatty acids.

It is generally believed that the p oxidation of fatty acids occurs in mitochondria in mammalian cells. The enzymes of /3 oxidation (acyl-CoA dehydrogenases, enoyl-CoA hydratase or “crotonase,” P-hydroxyacyl-CoA dehydrogenase, and thiol- ase) are thought to be localized exclusively in mitochondria. The results presented in this paper demonstrate that this is not the case. These enzymes are present in peroxisomes as well, where they catalyze the p oxidation of long chain fatty acids.

Recently Lazarow and de Duve (1) found that rat liver peroxisomes are capable of oxidizing palmitoyl-Cob, with the reduction of O2 to Hz02 and NAD to NADH. In the presence of CoA, several moles of NAD were reduced per mole of palmit- oyl-CoA added, and the reactions were uninhibited by 1 mM KCN. These data were consistent with a pathway of /3 oxidation, but in those initial studies we did not investigate whether the site of oxidation was indeed the p carbon, nor whether acetyl-CoA was in fact, the product. Therefore no conclusion was drawn concerning the exact mechanism.

In the present study, the spectroscopic techniques developed by Lynen and Ochoa (2) and by Mahler (3) in their classical

* This research was supported by National Science Foundation Grant PCM76-16657 and by National Institutes of Health Grants AM19394, HL20909, and RR07065. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “advertisement” in accord- ance with 18 U.S.C. Section 1734 solely to indicate this fact.

studies of mitochondrial /? oxidation were used to investigate whether or not purified peroxisomes are capable of carrying out the specific reactions of /3 oxidation. The appropriate 4- carbon acyl-CoA substrates (butyryl-CoA, crotonyl-CoA, and acetoacetyl-Cob) were employed, and the products were char- acterized by spectroscopic and enzymatic means. The suhcel- lular distribution of the enzymes of p oxidation was investi- gated with the techniques of analytical cell fractionation developed by de Duve and his collaborators (4). The results demonstrate that peroxisomes (as well as mitochondria) con- tain crotonase, /3-hydroxybutyryl-CoA dehydrogenase, and thiolase activity. Furthermore, peroxisomes catalyze the pro- duction of acetyl-CoA from palmitoyl-CoA. Therefore the peroxisomal fatty acid oxidation is a j3 oxidative process. The results obtained thus far suggest that the peroxisomes are a major site for the /3 oxidation of long chain fatty acids.’

MATERIALS AND METHODS

The spectroscopic assays we have used are based on those de- scribed by Lynen and Ochoa (2) and by Mahler (3). They make use of the characteristic absorption bands of the substrates and products of the reactions under investigation (Fig. 1). All of the acyl-CoA compounds of Fig. 1 absorb at 260 nm, due to the CoA itself, and in the vicinity of 233 nm, hue to the thioester linkage. In addition, crotonyl-CoA and acetoacetyl-CoA have their own characteristic absorption bands described below. A Cary 14, a Gilford 220 and a Gary 118 spectrophotometer were used in various parts of this investigation.

Crotonase was assayed by measuring the disappearance upon hydration (Fig. 1) of crotonyl-CoA’s specific absorption band at 263 nm. This absorption at 263 depends on both the double bond and the thioester linkage (2). The reaction mixture consisted of 50 nmol of crotonyl-CoA and 0.04% Triton X-100 in 1 ml of 25 rnM Tris/HCl buffer, pH 8.0, at 25”, unless indicated to the contrary in the figure legends. The change in A,,, was converted to nanomoles of crotonyl- CoA hydrated using AE = 6.4 mM-’ cm-’ (5).

P-Hydroxybutyryl-CoA dehydrogenase was assayed in the reverse direction from that shown in Fig. 1, by following the oxidation of NADH at 340 nm with acetoacetyl-CoA as substrate. The l-ml reaction mixture contained 50 PM acetoacetyl-CoA, 55 PM NADH, 0.01% Triton X-100, 12 mM dithiothreitol, 1 rnM KCN, and 25 rnM TrislHCl buffer, pH 8.0, at 25”. Ac = 6.22 rnM-’ cm-’ (5). Under these conditions there was some nonenzymatic hydrolysis of the substrate; subsequently I found that this may be alleviated by lowering the dithiothreitol concentration to 0.1 mM.

Thiolase was assayed by measuring the specific absorption band at 233 nm due to the thioester bond. Acetoacetyl-CoA was used as substrate and the increase inA,,, was followed as 1 nmol of substrate was converted to 2 nmol of acetyl-CoA. The reaction mixtures

’ These results were presented as a poster at the 11th FEBS Meeting in Copenhagen, August 14-19, 1977 (Lazarow, P. B. (1977) 11th FEBS Meeting, Abstract L-A-117).

1522

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Rat Liver Peroxisomes Catalyze Fatty Acid p Oxidation 1523

usually contained 50 nmol of acetoacetyl-CoA and 100 nmol of CoA in 1 ml of 2.5 rn~ Tris/HCl buffer, pH 8.0 at 25”. AC = 4.5 IIIM-’ cm-’ (5).

In some experiments acetoacetyl-CoA was detected by measuring a difference spectrum in the presence and absence of 10 mM MgCl,. Acetoacetyl-CoA in Tris buffer, pH 8, has a small absorbance band at 303 nm (Fig. 2, lower curue) due to formation of the enolate ion (pK = 8.5; Ref. 2). The intensity of this band is greatly increased by the addition of MgZ+ ions (Fig. 2, upper curue and inset), which probably form a chelate structure (2). Some attempts were made to make use of this effect in the routine assay of thiolase, but MgCl, was found to inhibit the reaction.

Acetyl-CoA was assayed by means of the citrate synthetase reaction coupled to the malate dehydrogenase reaction, essentially as described by Decker (6). The assay conditions were slightly modified to increase the sensitivity: we used a reaction volume of 1 ml containing 0.5 rnM nn-malate, 0.15 rnM NAD, and 0.2 M Tris/HCl, pH 8.1, at 25”. The sequential increases in A,,, upon addition of 0.02 unit of malate dehydrogenase and 0.17 unit of citrate synthetase were measured in a Cary 118 spectrophotometer. They served to calculate the amount of acetyl-CoA present, taking into account the nonlinear stoichiometry between acetyl-CoA reacted and NAD re- duced (6). This method was found to be accurate and reproducible when known amounts of acetyl-CoA were added directly to the assay mixture (solid circles in Fig. 31, and the assay is believed to be highly specific for acetyl-CoA (6).

fl Ctt-CH2-CH2-C-SCoA

I-- 2H Bufyryl-CoA dehydrogenase

0

CH3-CH=CH-!-SCoA

l- W’ Crotonose A &263 = -6.4

?H B CH3-CH-CH2-C-SCoA

F

NAD+ p- hydroxybutyryl-Co8

NADH + H+ dehydrogenose At& = 6.22

8 e CH3- C - CHZ - C - SCoA

l-

CoASH Thlolase A&233=45

0

2 CH,-%-SCoA

FIG. 1. Enzyme reactions investigated and specific absorption bands used. Changes in millimolar extinction coefficients are given. SCoA, coenzyme A in thioester linkage.

When this method was used to assay acetyl-CoA produced in reactions using peroxisomes, we first precipitated the peroxisomal proteins with cold perchloric acid, neutralized with KOH and KHCO,, and centrifuged down the KClO, and precipitated proteins. When known amounts of acetyl-CoA were carried through this procedure the assay was still linear, but on some occasions the recovery was less than 100% (Fig. 3, open circles).

It appears that the acetyl-CoA is not actually lost, but rather is underestimated in the assay due to incompletely removed perchlo- rate making the enzyme reactions sluggish. Perchlorate precipita- tion may be incomplete if the pH becomes slightly alkaline rather than remaining just under 7 during neutralization or if the samples are not kept ice-cold during centrifugation. As shown in the inset in Fig. 3, the supernatant after perchloric acid precipitation may interfere with the determination of acetyl-CoA assayed directly. In the experiments described in this paper, acetyl-CoA standards were always included and carried through the perchloric acid treatment, and in several cases, known amounts of acetyl-CoA were added to duplicate experimental aliquots.

The peroxisomes used in these experiments were purified by differential and equilibrium density centrifugation from the livers of three rats treated for 2 weeks with 0.5% cloiibrate in their food, as described previously (1). Cloilbrate was used because it increases the activity of the peroxisomal system of fatty acid oxidation by approximately 1 order of magnitude (1). Based on analysis of the marker enzymes, these peroxisomes were about 80% pure (data of Fig. 15, Table IV, and Ref. 7). The principal contaminant on a protein basis was microsomes, with smaller amounts of lysosomes and mitochondria. The enzymes under investigation were assayed in all the fractions from the equilibrium density centrifugation in order to verify that the enzyme activities measured in “purified peroxisomes” belonged to the peroxisomes themselves, and not to any of the contaminants.

Protein, catalase, cytochrome oxidase, glucose-6-phosphatase, and acid phosphatase were assayed according to Leighton et al. (7). Palmitoyl-CoA oxidation was assayed as described by Lazarow and de Duve (see legend to Fig. 6 of Ref. 1). Carnitine acetyltransferase was measured essentially according to Solberg et al. (8).

The following units are employed: protein (Lowry) is expressed as milligrams based on a bovine serum albumin standard. Catalase and cytochrome oxidase obey first order reaction kinetics: one unit of catalase decreases the H,O, concentration lo-fold/min at 0” in a volume of 50 ml (7); 1 unit of cytochrome oxidase oxidizes 90% of the cytochrome c/min at 25” in a volume of 100 ml (9). All other enzyme units are in micromoles/min. In the case of the palmitoyl-CoA oxidation assay this refers to micromoles of NAD reduced.

Acyl-CoAs were purchased from Sigma (St. Louis) or P-L Bio- chemicals (Milwaukee). Their concentrations were calculated by dividing the absorbance at 260 by a millimolar extinction coefficient of 16 (5) (or 22.6 for crotonyl-CoA) and multiplying by the stated purity of the manufacturer which varied between 79 and 91%. Cloiibrate was generously provided by Ayerst Laboratories (New York).

A

04. ~

o- MgCI2 fmMl

PI

+tdg++

0.2 - - btg++

200 L.

250 300 350 400

Wavelength (nm)

FIG. 2. Effect of 10 mM MgCl, on the absorption spectrum of acetoacetyl-CoA. The inset shows the effect of the Mg*+ concentra- tion on the magnitude of the A,,,.

Acetyl-CoA added lnmol)

FIG. 3. Assay of acetyl-CoA directly (0) and after perchloric acid U’CA) precipitation (0). The effect of added neutralized perchloric acid supernatant (without acetyl-CoA) on the determination of 10 nmol of acetyl-CoA measured directly is shown in the inset.

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1524 Rat Liver Peroxisomes Catalyze Fatty Acid p Oxidation

RESULTS

Crotonase - Peroxisomes catalyze a decrease in the absorp- tion characteristic of crotonyl-CoA (Fig. 4). The reaction stops with 21% of the crotonyl-CoA remaining, indicating that hydration, not hydrolysis, is the mechanism. This stoichiom- etry agrees with the value of 77% hydration that may be calculated from the equilibrium constant of 0.062 reported by Stern (10).

As illustrated in Fig. 5, the rate of the crotonase reaction is considerably increased by the inclusion of Triton X-100 in the reaction mixture. The rate of the reaction is proportional to the amount of peroxisomal protein added, both in the presence and absence of 0.04% Triton X-100 (Fig. 6). In the former case, the slope of the regression line fitted by least squares analysis gives a specific activity of 27 ymol of crotonyl-CoA hydrated/ min/mg of peroxisomal protein. The specific activity is 10 times lower in the absence of the detergent.

PHydroxybutyryl-CoA Dehydrogenase - As shown in Fig. 7, peroxisomes catalyze the oxidation of NADH with acetoace- tyl-CoA as substrate. The rate of the reaction is proportional to the amount of peroxisomal protein added (Fig. 8) and corresponds to a specific activity of 1.4 pmol/min/mg of perox- isomal protein.

Thiolase - Peroxisomes catalyze an increase in thioester bond absorbance (Fig. 9); no reaction occurs if either acetoace- tyl-CoA or CoA is omitted. The observed increase of 31 nmol of thioester linkage is quantitatively consistent with the expected doubling of acyl-CoA. In a duplicate reaction an increase of 32 nmol was observed.

The spectra measured in a subsequent experiment (Fig. 10) demonstrate both a decrease in the size of the small absorption band at 303 nm, characteristic of acetoacetyl-CoA, and the increase at 233 nm due to thioester bond formation. The disappearance of substrate during the reaction is shown more clearly by the Mg*+ difference spectra of Fig. 11. The formation of acetyl-CoA is given in Table I, together with the stoichiom- etry of the other changes observed. It is clear from these data that there was a nearly quantitative conversion of acetoacetyl- CoA to acetyl-CoA in a 1:2 ratio.

Fig. 12 illustrates that the rate of the reaction depends linearly on the amount of peroxisomes added. The specific activity is 2.8 pmol of acetoacetatelminlmg of peroxisomal protein.

AA263 -01

+ 0 4 Minutes

Linked Sequential Reactions-The results described thus far indicate that peroxisomes are capable of catalyzing each of the crotonase, P-hydroxybutyryl-CoA dehydrogenase, and thi- olase reactions. This conclusion was tested further by attempt- ing to link the three reactions to see whether the product of the first would serve as the substrate of the second, etc.

In the experiment illustrated in Fig. 13, 33 nmol of crotonyl- CoA were placed in a cuvette in 1 ml of 50 mM Tris/HCl buffer, pH 9.0, and the spectrum was measured (Curue A). Peroxi- somes were added to both sample and reference cuvettes, and the spectrum was remeasured (Curve B). There was a drop in the absorbance at 263 nm corresponding to the hydration of 28 nmol of crotonyl-CoA. NAD was then added to both cuvettes. Small absorption peaks appeared at 340 and 303 nm (Spectrum C), corresponding to the reduction of 5 nmol of NAD to NADH and the formation of 5 nmol of acetoacetyl-CoA. The reaction was incomplete; from the equilibrium constant of 6.3 x lo-” given by Wakil (11) one would expect to form 8 nmol of acetoacetyl-Cob. At this point CoA was added to both cu- vettes: formation of acetyl-CoA would make the overall path- way exergonic. As illustrated in Curve D, the absorption peak at 340 nm increased, corresponding to the reduction of a further 10 nmol of NAD to NADH. The peak at 303 nm vanished, indicating the disappearance of the acetoacetyl- CoA. The absorbance at 233 increased by an amount corre- sponding to the formation of 14 nmol of thioester bond, presumably by conversion of 14 nmol of acetoacetyl-CoA to 28

AA340 ffyy--J * ‘:j,7

0 2 4 0 2 4 6 Minutes Peroxisomal

protein (pg)

FIG. 7 (left). Time course of /3-hydroxybutyryl-CoA dehydrogen- ase reaction, assayed in reverse. The reaction was initiated by the addition of 6.9 +g of peroxisomal protein.

FIG. 8 (right). Effect of peroxisome concentration on the rate of the fi-hydroxybutyryl-CoA dehydrogenase reaction.

nmol xii

Triton X-100(%)

FIG. 4 (left). Time course of the hydration of crotonyl-CoA cata- rate of the crotonase reaction. Each reaction mixture contained 40 lyzed by 0.7 (A) or 1.4 pg (B) of peroxisomal protein. The base-line nmol of crotonyl-CoA and 1.4 pg of peroxisomal protein. is drawn at -0.245 A, the decrease observed upon alkaline hydroly- FIG. 6 (right). Effect of peroxisome concentration on the rate of sis of an identical aliquot of crotonyl-CoA, from which we calculate the crotonase reaction in the presence (x) and absence (0) of 0.04% that there were 38 nmol of substrate present. Triton X-100.

FIG. 5 (center). Effect of the Triton X-100 concentration on the

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A 05

010

AA 233

005

Mtnules

02

Rat Liver Peroxisomes Catalyze Fatty Acid j3 Oxidation 1525

O-

5-

o-

5-

250 300 Wavelength lnmi

FIG. 9 (left). Time course of the thiolase reaction. The reaction mixture contained 30 nmol of acetoacetyl-CoA, 100 nmol of CoA, and 17 pg of peroxisomal protein in 1 ml of 30 mM phosphate buffer, pH 7.4, at 37”.

FIG. 10 (center). Spectra before and after the thiolase reaction. The sample cuvette contained 47 PM acetoacetyl-CoA, 100 /AM CoA, and 25 rnM Tris/HCl buffer, pH 3.0. The reference cuvette contained CoA and Tris/HCl only. After measuring the spectrum, and remov- ing aliquots for later determination of acetyl-CoA and a MgZ+ difference spectrum, purified peroxisomes were added to both sample and reference cuvettes at a final concentration of 6.9 pg of peroxi-

TABLE I

Stoichiometry of thiolase reaction The reaction is described in the legend to Fig. 10. No acetyl-CoA

was detected in the reaction mixture before addition of peroxisomes nor in the peroxisomes themselves.

Component nmol/ml

Acetoacetyl-CoA added 47 Acetoacetyl-CoA after reaction 2 Thioester bond, net increase 44 Acetyl-CoA formed 91

nmol of acetyl-CoA. Spectrum D’ was taken 30 min later, when the reactions were complete; there was a net increase of 22 nmol of thioester bond and a reduction of a total of 26 nmol of NAD.

In order to show the transient accumulation of acetoacetyl- CoA more clearly, this experiment was repeated exactly as described, except that 10 times more NAD was added in order to shift the @-hydroxybutyryl-CoAJacetoacetyl-CoA equilib- rium forward. This, however, prevented measuring spectra below 280 nm, due to the intense absorption of the NAD. As illustrated in the lower part of Fig. 13, Spectrum C, use of this larger amount of NAD considerably increased the sire of the absorption peaks at 303 and 340 nm. They correspond to the accumulation of 11 nmol of acetoacetyl-CoA and the reduction of 12 nmol of NAD, respectively. Addition of CoA again resulted in the disappearance of the acetoacetyl-CoA and further reduction of the NAD. As expected, the total NADH formation (29 nmol, Curve D’) was similar to that observed in the first experiment.

These experiments were repeated several times, taking a number of spectra at each step, and generally similar results were obtained. However, the final yield of thioester bond formed is lower when many spectra are taken at step C, due to gradual hydrolysis of acetoacetyl-CoA which occurs at the

AA

Wovelength inm)

somal protein/ml. The reaction was followed at 233 nm; when it was complete, the second spectrum was taken.

FIG. 11 (right). Mg*+ difference spectra before and after the thiolase reaction to test for acetoacetyl-CoA. Duplicate aliquots of the reaction mixture (before addition of peroxisomes) were placed in sample and reference cuvettes. Their difference spectrum was every- where zero as expected. Then 10 rnM MgClz was added to the sample cuvette, 1 mM EDTA was added to the reference cuvette and the difference spectrum was measured (top curve). A similar differ- ence spectrum was measured on duplicate aliquots of the reaction mixture after the thiolase reaction was over (bottom curve).

m min

I , b

5 IO 15 Peroxisomol protein (pg)

FIG. 12. Effect of peroxisome concentration on the rate of the thioiase reaction. Reaction conditions as in Fig. 10.

alkaline pH of 9 that was employed. Hydrolysis may be reduced by working at pH 8, but this shfis the equilibrium of the /3-hydroxybutyryl-CoA dehydrogenase reaction further away from acetoacetyl-CoA.

Butyryl-CoA Dehydrogenase - Since crotonyl-CoA serves as substrate for the linked reactions leading to acetyl-CoA, we made use of this fact to assay for the desaturation of butyryl- CoA (Fig. 1, first reaction). Butyryl-CoA was added to a reaction mixture containing peroxisomes, NAD, and CoA (Table II) and the formation of NADH was measured spectro- photometrically at 340 nm. No reaction was detected. Addition of crotonyl-CoA demonstrated that the assay system was functioning correctly.

Oxidation ofPalmitoyl-CoA to Acetyl-CoA -A l-ml reaction mixture was prepared containing 10 nmol of palmitoyl-CoA together with excess NAD and CoA. The addition of peroxi- somes resulted in the reduction of 52 nmol of NAD to NADH

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1526 Rat Liver Peroxisomes Catalyze Fatty Acid /3 Oxidation

TABLE II

Coupled assay for butyryl-CoA dehydrogenase Reactions contained 10 nmol of substrate and 8.6 pg of peroxi-

somal nrotein: other conditions as in Table III.

A Crotonyl-CoA

B +Peroxlsomes

C +NAD D +CoA

Substrate

Butyryl-CoA Crotonyl-CoA

NAD reduction

Initial rate Extent nmollmin n?nol

0 0 4.0 8.5

TABLE III

Acetyl-CoA formed by peroxisomes from palmitoyl-CoA

Reaction mixture” Palmito l- CoA ad B ed “Eel-

Ace;&C~A

nmol nmol ?lOZOl

Complete Experiment 1 10 52 50 2 1

0 0 0 Experiment 2 10 48 48 + 2

0 0 0 Peroxisomes 10 0 0

omitted

250

Wavelength (nm)

FIG. 13. Sequential p oxidation reactions. Upper graph: A, spec- trum of crotonyl-CoA (33 nmol by alkaline hydrolysis) in 1 ml of 50 mu Tris/HCl buffer, pH 9.0, at 25”. The reference cuvette contained buffer only; B, spectrum after addition of 34 pg of peroxisomal protein to both sample and reference cuvettes; C, spectrum after subsequent addition of 50 nmol of NAD to both cuvettes; D, spectrum after subsequent addition of 50 nmol of CoA to both cuvettes; D’, spectrum 30 min later. Lower graph: identical experiment except that 500 nmol of NAD were added to the sample cuvette only before Spectrum C. Curves A and B (not shown) were very similar to those in the upper graph. These spectra were measured with a Cary 118 spectrophotometer using a constant slit width of 0.7 mm, automatic control of the gain to maintain constant signal energy and a scan speed of 1 rim/s. Identical spectra were observed with narrower slits or slower scan speeds. The scans were started 4 min apart except for D and D’ which followed their predecessors by 8 and 30 min, respectively. Calculations of metabolite concentrations (see text) take into account dilutions of 2 or 2.5% at each step.

(Table III). No NAD reduction occurred in control reaction mixtures in which palmitoyl-CoA or peroxisomes were omit- ted. The reaction mixtures were assayed for acetyl-CoA: 50 nmol of acetyl-CoA were formed in the 1 ml of complete mixture with substrate (and none in the controls) (Table III). This is in good agreement with the value of 52 nmol of NAD reduced and indicates that the 10 nmol of palmitoyl-CoA went through five cycles of /3 oxidation. The experiment was repeated a second time with very similar results (Table III).

Chain Length SpecifEity of Peroxisomal Fatty Acid Oxida- tion - The ability of peroxisomes to oxidize fatty acyl-CoAs of various chain lengths was determined by measuring the rate of NAD reduction. This assay involves the concerted action of all four /3 oxidation enzymes. As shown in Fig. 14, peroxisomes appear to have similar activity toward lauroyl- and palmitoyl- CoA, but the activity is considerably lower with octanoyl-

D Complete reaction mixtures contained 100 nmol of CoA, 200 nmol of NAD, and 34 pg of peroxisomal protein in 1.00 ml of 50 mM TrislHCl buffer, pH 8.0, with 1 mM dithiothreitol, 0.01% Triton X- 100 and 0.0075% bovine serum albumin, at 37”.

* Acetyl-CoA was determined on quadruplicate aliquots of each reaction mixture and corrected for recovery of standard acetyl-CoA added to half of the aliquots; the recovery was 75% in the first experiment and 101% in the second. Means and standard deviations are indicated.

Chain length

FIG. 14. Ability of peroxisomes to oxidize acyl-CoAs of varying chain lengths. Reaction mixtures contained 10 nmol of substrate and 9.6 pg of peroxisomal protein; other conditions were as described previously (l), including 0.1 mM CoA. Means and ranges of duplicate determinations are indicated.

CoA and no activity was detected with butyryl-CoA as sub- strate. Thus the peroxisomes appear specialized for the oxida- tion of longer chain length fatty acids.

Distribution of p Oxidation Enzymes after Equilibrium

Density Centrifugation - The activities of the various enzymes were assayed in all the fractions from the isopycnic centrifu- gation experiment by which the purified peroxisomes used in these studies were prepared. The results are shown in Fig. 15, together with the distributions of catalase and cytochrome oxidase, which are used as marker enzymes for peroxisomes and mitochondria, respectively. Fig. 15 also includes the distributions of protein, as well as of glucose-6-phosphatase

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Rat Liver Peroxisomes Catalyze Fatty Acid /3 Oxidation 1527

600

400

200

60

40

20

60

40

20

h

oc

Crotonose

Thiolose

I( Hydroxybutyryl-CoA

Cotolose /

Cytochrome oxldose (mrtochondriol 1

Density I ’

20 DISCUSSION

Acid phosphotose (lysosomes) /

Glucose-6-phosphotose (microsomes) I

Volume (ml) FIG. 15. Distribution of enzyme activities after equilibrium den-

sity centrifugation of the peroxisome-rich fraction described in Table IV. The ordinate scales are chosen so that the areas under all the histograms are the same, in effect normalizing the distributions to facilitate their comparison (4). The peak peroxisomal fraction was used for all the other experiments described in this paper.

and acid phosphatase, marker enzymes for microsomes and lysosomes, respectively, which were present in small quanti- ties in the peroxisome-rich fraction layered on the gradient (Table IV). It may be emphasized that this layer was much poorer in mitochondria than in peroxisomes (Table IV).

Crotonase, /3-hydroxybutyryl-CoA dehydrogenase, and thi- olase each have a major peak of activity similar in shape and position to that of catalase, demonstrating that these enzymes are present in peroxisomes. In addition, they each have a shoulder or smaller peak in the center of the gradient, where the mitochondria are localized, as shown by the distribution of cytochrome oxidase.

Fig. 15 also shows the distribution of carnitine acetyltrans- ferase, which is present in similar concentrations in the

TABLE IV

Composition of peroxisome-rich fraction prepared by differential centrifugation from livers ofclofibrate-treated rats

Marker for Homage- Peroxisome-rich nate fraction”

Protein 217 9.6 4.4 Catalase Peroxisomes 125 13.7 11 Cytochrome oxidase Mitochondria 33.5 0.93 2.8 Glucose-6-phospha- Endoplasmic 19.3 1.2 6.2

tase reticulum Acid phosphatase Lysosomes 9.5 1.3 14

a This fraction was subjected to equilibrium density centrifuga- tion in the experiment illustrated in Fig. 15.

* Units are defined under “Materials and Methods.” The starting material was 35 g of liver.

peroxisomal and mitochondrial fractions. Lastly, the distribu- tion of palmitoyl-CoA oxidizing activity (measured as the rate of NAD reduction (1)) is included. This appears to be virtually identical with that of catalase; no additional activity in the region of the mitochondria is evident.

The peak peroxisomal fraction of Fig. 15 was used in all the other experiments described previously.

The results demonstrate that hepatic peroxisomes of clofi- brate-treated rats contain crotonase, p-hydroxybutyryl-CoA dehydrogenase, and thiolase. Furthermore, the peroxisomes convert palmitoyl-CoA to acetyl-CoA. Therefore, the peroxi- somal fatty acid-oxidizing system reported previously (1) pro- ceeds by a mechanism of p oxidation.

It should be emphasized that although these experiments were performed on peroxisomes from rats that had been treated with cloiibrate in order to increase the activity of their peroxisomal system of fatty acid oxidation (11, hepatic peroxisomes from normal rata also contain these three en- zymes, although at much lower levels.Z Thus clotibrate affects the activity, but not the basic mechanism, of this peroxisomal enzyme system.

Butyryl-CoA desaturation activity was not detected in these purified peroxisomes. Beinert, in his review of (mitochondrial) acyl-CoA dehydrogenases (12), states that most tissues studied have two (and pig liver three) acyl-CoA dehydrogenases, active on short, (medium), and long chain acyl-CoAs. The other three enzymes of p oxidation appear to be active on all the different chain length substrates that have been tested (10, 11, 13). These facts, together with the observation that peroxisomes reduce 0, to H,O, in the presence of palmitoyl- CoA (1, 14) lead us to infer that peroxisomes contain an enzyme capable of the cr,p desaturation of long chain acyl- CoAs (although it appears to function as an oxidase rather than a dehydrogenase), but have little or no enzyme active on short chain acyl-CoAs (which however is found in mitochon- dria2).

In our first experiments only three cycles of p oxidation were observed with palmitoyl-CoA as substrate (1). After reiining the reaction conditions, five cycles have now been detected. The apparent inactivity of the peroxisomes toward short acyl-CoAs provides an explanation for the fact that we have not observed the theoretical maximum of seven cycles.

Acetyl-CoA was observed to accumulate as the end product * P. B. Laxarow, unpublished results.

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1528 Rat Liver Peroxisomes Catalyze Fatty Acid /3 Oxidation

of hepatic peroxisomal /3 oxidation in these experiments. This differs from previous studies by other investigators with other organelles or tissues, in which the acetyl-CoA was further metabolized to ketone bodies, to intermediates of the Krebs or glyoxylate cycles, or to CO,. The fate of the acetyl- CoA produced in the peroxisomes is not known. It may be transported to the mitochondria for further oxidation or used for biosynthetic reactions elsewhere in the cell.

Carnitine may play a role in transporting this “active acetate” to the mitochondria, which are believed to be im- permeable to acetyl-CoA (15). The presence of carnitine ace- tyltransferase in both peroxisomes and mitochondria was first reported by Markwell et al. (16). Solberg et al. (8) and Moody and Reddy (17) found that the activity of this enzyme is increased in the livers of rats treated with clotibrate, such as the rats used in these investigations. The present experiments confirm the presence of carnitine acetyltransferase in both peroxisomes and in mitochondria. Furthermore they provide a plausible explanation for the presence of this enzyme in peroxisomes by showing that peroxisomes actively synthesize acetyl-CoA, the enzyme’s substrate. We infer that carnitine functions as an interorganellar shuttle of acetyl (and perhaps other acyl) residues.

Until now it has been generally believed that the p oxida- tion of fatty acids is a mitochondrial function in mammalian cells. The observation that peroxisomes catalyze /3 oxidation raises the questions: Is there any /3 oxidation in mitochondria? Was peroxisomal /3 oxidation attributed to mitochondria be- cause mitochondrial fractions prepared by differential centrif- ugation are contaminated by peroxisomes? The fractionation data of Fig. 15 provide some clues. There are major peaks of crotonase, thiolase, and P-hydroxybutyryl-CoA dehydrogen- ase in peroxisomes, and small peaks or shoulders of these enzymes where the mitochondria are located. However, since the starting material layered on this gradient was consider- ably enriched in peroxisomes relative to mitochondria, I calculate that in the cell both organelles may have roughly similar amounts of these enzymes. Further investigations of their subcellular distributions are in progress. The impression one gets from Fig. 15 that in clofibrate-treated rats, most of the hepatic palmitoyl-CoA oxidation occurs in peroxisomes has received considerable additional experimental support; even in normal rats, the peroxisomes make a substantial con- tribution to palmitoyl-CoA oxidation.3 The data available so far suggest that both peroxisomes and mitochondria cata- lyze p oxidation, but their specificities are not the same.

The finding that rat liver peroxisomes oxidize fatty acids provides a function of obvious physiological importance for the mammalian peroxisomes. Moreover, it demonstrates a close functional kinship between peroxisomes in a wide vari- ety of cell types, including plants and protozoa. Ten years ago, Breidenbach and Beevers (18) reported that germinating fatty seedlings contain an organelle with all the enzymes of the glyoxylate cycle, which they termed the “glyoxysome.” Subsequently, Cooper and Beevers (19) found that this organ- elle catalyzes /3 oxidation. Glyoxysomes also contain catalase

3 P. B. Lazarow, manuscript in preparation.

and oxidases, making them a member of the peroxisome family as defined by de Duve and Baudhuin (20). Blum (21) has reported that Tetruhymena peroxisomes contain some enzymes of p oxidation, and Graves and Becker (22) have provided similar evidence for the glyoxysomes of Euglena. These facts support de Duve’s speculations concerning a com- mon ancient evolutionary origin for animal and plant peroxi- somes and glyoxysomes (23).

In addition to its physiological function, the hepatic peroxi- somal system of p oxidation appears to play a role in reducing serum lipid levels during therapy with hypolipidemic drugs. Three different drugs greatly increase hepatic palmitoyl-CoA oxidation in the rat, at least in part by enhancing the activity of this peroxisomal enzyme system (24).

Acknowledgments-1 would like to thank Ms. Annabella Bushra for her expert and dedicated technical assistance, and Dr. C. de Duve for his helpful criticism of this manuscript.

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P B LazarowRat liver peroxisomes catalyze the beta oxidation of fatty acids.

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