In, J Bmhum, Vol. 12, pp 625 to 630 0 Pergamon Press Ltd 1980 Prmted m Great Bntam
FATTY
W?O-71
I X/XO,WOl-0625fO2.00/0
ACID PRODUCTS OF PEROXISOMAL j?-OXIDATION
H. OSMUNDSEN,C. ELIZABETHNEAT and B. BORREBAEK Institute for Medical Biochemistry, University of Oslo, Box 1112, Blindern, Oslo 3, Norway (Received 20 March 1980) Abstract-l. Fatty acid products from the peroxisomal oxidation of [U-“‘C]palmitoyl-CoA and [10-‘4C]oleoyl-CoA have been detected. Products of the first 2-3 b-oxidation cycles predominate. 2. Although NAD is associated with peroxisomal fatty acid oxidation as an obligatory cofactor, it is shown that also NADP can support some peroxisomal oxidation with these two acyl-CoA esters. 3. Peroxisomal preparations have been found to contain appreciable palmitoyl-CoA hydrolase (EC 3.1.2.-) activity, as well as hydrolase activities towards other acyl-CoA esters. Unlike b-oxidation, peroxisomal hydrolase activities are not influenced by clofibrate treatment. A possible role of these hydrolases in controlling the extent of peroxisomal acyl-CoA oxidation is discussed.
INTRODUCTION Rat liver peroxisomes have been shown to be able to p-oxidize acyl-CoA esters (Lazarow & de Duve, 1976). Peroxisomal /I-oxidation is markedly stimulated by clofibrate administration (Lazarow & de Duve, 1976; Lazarow, 1977). The peroxisomal oxidation of palmitoyl-CoA appears incomplete, involving at the most 5 P-oxidation cycles (Lazarow. 1977). With [14-14C] erucoyl-CoA we have found that the products from the first three b-oxidation cycles constitute about 9004 of fatty acid intermediates, the remaining 10% originating from the fourth cycle (Osmundsen et al., 1979). We have previously also found appreciable oxidation of erucoyl-CoA in the presence of NADP, although maximal oxidation was achieved in the combined presence of NAD and NADP (Osmundsen et al., 1979). We here report results obtained from studies of peroxisomal oxidation of [ l-14C] palmitoyl-CoA, [U-‘4C]palmitoyl-CoA and [ 10-14C]oleoyl-CoA. On clofibrate administration rat liver acyl-CoA hydrolase (EC 3.1.2.-) activities are increased markedly (Borrebaek et al., 1979). We have therefore examined peroxisomal preparations for acyl-CoA hydrolase activities.
otherwise as CoA esters
METHODS
diets Male Wistar which had been fed a dard pelleted laboratory diet supplemented 0.3”/, clofibrate weeks were used throughout study. The rats had been purchased
AmerBucks., U.K. The corresponding acyl-CoA esters were synthesized essentially as described (Al-Ahrif & Blecher. 1969). and purity et al.. NAD (grade were purchased Sigma ChemiCo., St MO, U.S.A. reagents used 625
et
1979). Cold (Shulz, 1974).
of isolated Rat livers homogenized in medium containing mM EGTA mM mannitol, aminoethylether) N, acid), 10 Hepes (N-2-hydroxyerhylpiperazine-N’2-ethanesulphonic acid), (w/v) homogenate. liver peroxisomes 42-51% sucrose gradient using the Sorvall OTD-65 ultra-centrifuge. Centrifugation were carried out by the TV-850 rotor. The details of the isolation procedure have been & Osmundsen, 1979). The peroxisomes within 4 hr of the animal being killed. Measurement of perovisomal P-oxidation Peroxisomal
1977)
increasing were identical
those described
(Osmunsen
with assay et al., 1979),
1979). Analysis
MATERIALS AND
(Osmundsen synthesized as
fatty acid o.uidation intermediates
those described above) were removed at various time intervals. total incubation volume was 3.5 ml, and usually contained 1.5-2.0 added to 0.5 ml of 5 M KOH (in 50X of methanol).,. ,_., and heated for 30 min at 90°C. After having been cooled on ice, the samples were acidified with HCI (ice cold) and extracted twice with 5 ml of diethylether. To the pooled etherphase was subsequently added 20~1 1 M KOH (in ethanol), and blown to dryness with a stream of N,. Radiog.1.c. analysis was carried out by using a Pye 104 gas chromatograph connected to ES1 Nuclear radioactivity detector with a I: 1 outlet splitter. The radioactivity in the various peaks was quantitated by using an ES1 Nuclear printing autoscaler (model 5680). The dry samples were immediately before injection, solubilised by the addition of 30 pl of 4 N HCI in dioxane’ (product of Pierce, Rockford, IL, U.S.A.). while the samples
626
H.
OSMUNDSEN.
C.
ELIZABETH NEAT and B. BORREBAEK
greater than it is from [U-‘4C]paimitoyl-CoA. After 15 min of incubation about 90”/, of added radioactivity from [l- 14C]palmitoyl-CoA has appeared as soluble radioactivity. In contrast, only about 10% of added [U-“Clpalmitoyl-CoA radioactivity has become acid soluble. &J&s oJ NAD and N ADP on pero.~iso~ial o.~idatioil oj’[U-‘“Cl 60
t
palmitoyl-CoA and [lU-r4C]ole~~I-C~A
The data presented
in Fig. 2 show that NADP alone will support only minimal peroxisomal with [U-‘4CJpalmitoyl-CoA. With /Goxidation [IO-*4C]oieoyl-CoA as substrate the extent of NADP supported oxidation resembles that obtained with [i4-“‘Clerucoyl CoA (Osmundsen rt al., 1979). Further, when present together with NAD (in equimolar concentrations) NADP has no effect on the NAD-supported P-oxidation of [ 10-L4C]oleoyl-CoA, although an inhibition probably results with [U-‘4C]palmitoyl as substrate.
/I
Productsfrom pero.uisomal oxidation of [U-l ’ Clpalmitoy1 CoA and [10-14C]oleoyl-CoA. 10
5
10
ttmefmlnl
15
of [1-‘4C]palmitoyl-CoA and Fig. 1. Oxidation peroxisomes. [U-‘4C]palmitoyl-CoA by isolated 60 pM[ l-‘4C]palmitoyl-CoA (specific activity 260 dis~min/ nmol) and 60 ~M[U-‘4C]palmitoyl-CoA (specific activity 430 dis/min/nmol) were incubated separately with 0.6 mg of peroxisomal protein in a final volume of 1 ml. Samples (0.2 ml) were removed at the times indicated. and acid soluble radioactivity measured (here expressed as percentage of added radioactivity). The plotted data represent the mean values of two separate experiments, with SEM indicated. Experimental details are otherwise given in the Methods section.
were being kept on ice. Samples of 10 ~1 were injected into the gas-chromatograph. Free fatty acids containing 2-18 carbon atoms were conveniently separated on a column (1.8m, 4 mm internal diameter) of lOy;SP-216-PS on Supelcoport 100/120 (Supelco Inc. Bellefonte, PA, U.S.A.) using a temperature program from 10%195”C. increasing by 7.YC per min. The detector temperature was 23O”C, and the injector temperature about 110°C. Assay qf‘acyl-CoA hpdrolaseactiWes Acyl-CoA hydrolase activities were assayed spectronhotometricallv at 324nm in 20mM K,HPO,. DH 7.2, ising 0.5 mM pi-(4-dipyridyI~disulphide to follow COA-SH released. Each assay contained 0.1-0.3 mg of peroxisomai protein in a final volume of 1.0 ml. All assays were carried out at 37°C using a Beckman model 25 spectrophotometer. Assaysof’protein Proteins were assayed by using the Bio-Rad protein assay kit (Bio-Rad Laboratories, Richmond, CA, U.S.A.).
Results presented in Fig. 3 show that the products accumulating from the peroxisomal oxidation of [U-‘“Clpalmitoyl-CoA are mainly chain shor?ened fatty acid resulting from the first two fi-oxidation cycles. In addition a small amount of the product from the third oxidation cycle was detected (C,,:,) (about 107; of injected radioactivity). As expected a steady build up of radioactive acetate was also observed (see Fig. 3). With [IO-*4C]oleoyl-CoA. the intermediates accumulating were the products of the first 3 cycles of /I-oxidation (C16:,, C,,:,, C,,:,), although a small amount of C,O,O (resulting from the fourth ~-oxidation cycle) was also observed (at the most 5% of injected radioactivity) (see Fig. 3). A small, but steadily increasing amount of radioactive acetate was also detected. indicating that a small amount of [10-‘4C]oleoyl-CoA was oxidized beyond four cycles of ~-oxidation. Peroxisornal acyl-Cod hydrolase actioities Acyl-CoA hydrolase activities towards some acylCoA esters were measured in peroxisomal preparations from both clofibrate treated rats, and control rats. The data resulting from this investigation is presented in Table 2. The apparent K, for palmitoylCoA and acetyl-CoA were determined by using program Hyper (Cleland, 1963) run on a PDP 11/03 computer. The specific activity obtained with palmitoyl-CoA (about 79 nmol/min per mg of peroxisomal protein) is about S-fold higher than values obtained with mitochondrial fractions and about 2-fold higher than values for microsomal fractions (Berge & Dglssland, 1979). This suggest that the activity present in our peroxisomal fractions is a real peroxisomal activity. The purity of the peroxisomal fractions used (about 9%; peroxisomal protein, see Neat & Osmundsen, 1979) supports this argument.
RESULTS Rates of’ oxidation of [l-‘4~palmitoyl-CoA [U-“(T) pa~~~itoyi-CoA
and
The data presented in Fig. 1 show that the rate of of acid soluble radioactivity from appearance [ l-14C]palmitoyl-CoA is one order of magnitude
DISCUSSION
Pero.yisoma~~-o.~jdat~onof [l-‘4qpalmitoy~-CoA [ U-I4flpalmitoyl-CoA
and
The results presented in Fig. 1 showing oxidation of [1-‘4C]palmitoyl-CoA and [U-14C]palmitoyl-CoA is
621
Fatty acid products of peroxisomal /Soxidation
5
10 tlmeimln)
I
I
5
10
tlmeImln)
15
20
I
I
15
20
Fig. 2. The effects of NAD and NADP on peroxisomal oxidation of palmitoyl-CoA and oleoyl-CoA. 60 ~M[U-‘4C]palmitoyl-CoA (a) and 60~M[IO-‘4C]oleoyl-CoA (specific activity 610 dis/min/nmol) (b) were incubated in the presence of 0.5 mM-NADP (A), in the presence of 0.5 mM NAD and 0.5 mM NADP (0) and in the presence of 0.5 mM NAD (0). Release of acid soluble radioactivity was measured (here expressed as percentage of added radioactivity) at the times indicated. Each 1ml incubation contained 0.5 mg of peroxisomal protein. These data are derived from one peroxisomal preparation. The same patterns were obtained with two other peroxisomal preparations, although the extent of oxidation varied by a factor of 2. Other experimental details are given in the legend to Fig. I.
a simple illustration
of the incomplete peroxisomal /?-oxidation of palmitoyl-CoA. The ease by which the [l-14C]carbon atom from palmitoyl-CoA becomes acid soluble also suggests that care is required when measuring cellular fatty acid oxidation from l-14Clabeled palmitate, if this is to be a measure of the
amount of palmitate oxidized. This applies particularly when induction of peroxisomal /?-oxidation has taken place. For these reasons. and to enable analysis of /I-oxidation intermediates we have carried out all subsequent work using [U-14C]palmitoyl-CoA. The effect of NAD and NADP on peroxisomal oxidation of [U-‘“Cjpalmitoyl-CoA and [lo-“CjoleoylCoA
Peroxisomal j&oxidation has been shown to be obligatory dependent on NAD, due to the NAD requiring /3-hydroxyacyl-CoA dehydrogenase (EC 1.1.1.35) reaction. NADP has never been shown to be involved in any /?-oxidation sequence. Recently, how-
ever, we have found that appreciable peroxisomal oxi-
dation of [14-14C]eroucoyl-CoA can take place in presence of NADP alone (Osmundsen et al., 1979), and that the NAD-dependent oxidation of [14-r4C]erucoyl-CoA may be further stimulated by added NADP. These findings suggested a possible role of NADP, and it was thought to be of interest to see whether a similar NADP effect also is expressed with two shorter acyl-CoA esters. Results obtained with [U-14C]oleoyl-CoA and [10-14C]oleoyl-CoA suggest that NADP (at equimolar concentration have no marked effect on the NAD-dependent peroxisomal oxidation of these substrates (see Fig. 2). With [U-“Clpalmitoyl-CoA the extent of oxidation obtained in the presence of NADP alone was about 20”%of that obtained in the presence of NADP alone. With [10-14C]oleoyl-CoA, however, this was about 60%, and therefore similar to that obtained previously with [14-14C]erucoyl-CoA (Osmundsen et al., 1979).
628
H.
OSMUNDSEN,~. ELIZABETH NEAT and B. BORREBAEK
Table 1. The effect of acyl-carbon chain length on stoicheiometry of NAD reduction caused by defined amounts of various acyl-CoA species Acyl-CoA Erucoyl-
GadeoylPalmitoylDecanoyl-
NADH generated (nmol)
nmol added 3 5 9 5.5 II 2.5 5 IO 8 IO I6
NADH:acyl-CoA
IO 17 26 I6 32 5 II I9 I4 19 26
3.3 3.4 2.9 2.9 2.9 2.0 2.2 I.9 I.8 1.9 1.6
3.2 k 0.3
2.1 * 0.2 1.7 + 0.3
These results were obtained by using up to three different peroxisomal preparations. Acyl-CoA dependent NAD reduction was measured spectrophotometrically as described in the Experimental section. Mean values with sd are listed to the right hand side of the table.
:oo
10
5
I
b
time
(mm)
15
20
I
>i$;: 5
10
tfme
(mm)
15
20
Fig. 3. Products from peroxisomal oxidation of [U-‘4C]palmitoyl CoA and [10-‘4C]oleoyl-CoA. Peroxisomes (1.7 mg of protein) were incubated with 60 ~M[U-‘4C]palmitoyl-CoA (a) or [10-‘4C]oleoyl-CoA (b) in a final volume of 3.5 ml, as described in the Methods section. Samples were removed at the times indicated for analysis of fatty acids by radio-g.1.c. The amount of each component has been expressed as percentage of injected radioactivity. The percentage contribution of each species has been corrected for differences due to carbon chain lengths (applies to products from [U-r4C]palmitoyl-CoA only). The data presented were obtained in single experiments, but they are representative of results obtained in two other experiments. The carbon chain lengths of the fatty acid products are indicated in the figure. Acetate is indicated by (C,).
Fatty acid products of peroxisomal /l-oxidation
629
Table 2. Acyl-CoA hydrolase activities of peroxisomes isolated from clofibrate treated rats
Substrate Erucoyl-CoA Palmitoyl-CoA Decanoyl-CoA Acetyl-CoA
Concentration (mM) 0.050 0.130 0.10 0.06 1.40 0.16
Hydrolase activity of clofibrate treated rats (nmols/min/mg) 9.0(1.4) 6.9 (0.3) 79.3 (5.0) 6.4 (0.7) 8.1 (1.3) 7.5 (0.8)
Hydrolase activity of control rats (nmols/min/mg)
Apparent K, (M)
3.4 I (0.8) 78.9 (3.3)
1.5 x 10-s
5.45 (I .25) 5.83(1.0)
2 X 10-4
The tabulated values represent the mean of four observations with se indicated in parentheses. The apparent I<, for palmitoyl-CoA hydrolase was estimated from velocities measured at 8 different substrate concentrations, and that of acetyl-CoA hydrolase from velocities measured at 4 different substrate concentrations. It is tempting to speculate as to any Possible role of NADP as regards peroxisomal /3-oxidation. It is in our view, possible that it could have a regulatory
function in potentiating
oxidation of very long chain
acyl-CoA esters, while the oxidation of shorter acylCoA esters is inhibited. Thus NADP may have a vec-
torial effect in directing these shorter fatty acids to mitochondrial j?-oxidation. We have recently found evidence suggesting that CoA may have a similar function (Osmundsen & Neat, 1979). The data presented in Table 1 show that there is a trend for peroxisomal @oxidation to carry out more extensive chain shortening as the carbon chain lengths are increased from 10 to 22 carbon atoms. These findings, which are based on the stoicheiometries of NAD reduction are therefore in agreement with the above discussed analysis of accumulated products (see Fig. 3). This can be taken to show that peroxisomal oxidation may be of more relevance to very long chain fatty acids. The decrease in chain length from 22 to 18 carbon atoms would render the resultant mono-unsaturated acid an appreciably better substrate for mitochondrial @-oxidation (Osmundsen & Bremer, 1978). This argument would of course not apply to fatty acid containing 16 carbon atoms or less. Acyl-CoA
hydrolases of peroxisomal preparations It is apparent from the data of Table 2 that peroxisomes isolated from both control and clofibrate treated rats are about 10 times more active in the hydrolysis of palmitoyl-CoA than in the hydrolysis of any of the other acyl-CoA esters tested. It is also noteworthy that acetyl-CoA hydrolase was found in the peroxisomal preparations. The apparent K, for the palmitoyl-CoA hydrolase (15 PM) is one of the same order as that of the palmitoyl-CoA hydrolases present in other sub-cellular fractions (Kurooka et al., 1972). We have previously shown that acyl-CoA hydrolase activities, parti~larIy in rat iiver soluble supernatant, increase with clofibrate treatment (Borrebaek et af., 1979). It was therefore surprising to find that acylCoA hydrolase in peroxisomal preparations (in terms of specific activities) does not change on clofibrate treatment. This is in marked contrast to peroxisomal /?-oxidation (Lazarow & de Duve, 1976). The functional roles of these enzymes are not clear. In the peroxisomal context it is tempting to speculate that they may be involved in controlling the extent of oxidation. The palmitoyl-CoA and acyl-CoA
decanoyi-CoA hydrolase activities could explain why erucoyl-CoA (Osmundsen er al., 1979) and oleoylCoA are shortened primarily down to C,4 and C, Z fatty acids and palmitoyl-CoA down to Cl0 fatty acids (see Fig. 3). The peroxisomal decanoyl-CoA hydrolase activity is relatively low, but the particle free liver supernatant from clofibrate treated rats has a considerable amount of this activity (Borrebaek et a!., 1979). For shorter acyl-CoA esters (e.g. decanoylCoA) the low activity of peroxisomal /?-oxidation towards short chain acyl-CoA esters may also be a limiting factor.
Acknowledgemenrs-This work was supported by The Royal Norwegian Council for Scientific and Industrial Research. We thank Mr L. Bohne for excellent technical assistance.
REFERENCES AL-AHRIFA. & BLECHERM. (1969) Synthesis of fatty acylCoA and other thiol esters using Iv-hydroxysuccinimide esters of fattv acids. J. Lipid Res. 10. 344-345. BERGEK. R. & D$SSLAND’B.(1979) Differences between microsomal and mitochondrial-matrix palmitoyl-CoA hydrolase, and palmitoyl-carnitine hydroiase from rat liver. Biochem. J. 181, 119-125. BORREBAEKB., OSMUNDSENH. & BREMER J. (1979) Increased levels of hepatic acyl-CoA hydrolase activities in clofibrate fed rats. ARCS Med. Sci. 7, 181. CLELANDW. W. (1963) Computer programmes for processing enzyme kinetic data. Narure, Land. 198,463-465. KURDOKA S., HO~KI K. & YO~IMURA Y. (1972) Some properties of long chain fatty acyl-CoA thioesterase in rat organs. J. Biochem. Tokyo 71, 625-634. LAZAROWP. B. & DUVE C. DE (1976) A fatty acyl-CoA oxidizing system in rat liver peroxisomes; enhancement by clofibrate, a hypolipideamic drug. Proc. natn. .&ad. Sci. U.S.A. 73, 2043-2046.
LAZAROWP. B. (1977)Three hypolipideamic drugs increase hepatic palmitoyl-CoA oxidation in the rat. Science 197, 58G581.
LAZAROWP. B. (1978) Rat liver peroxisomes catalyze the j%oxidation of fatty acids J. hioi. Chem. 253. t 522- 1528. NEAT C. E. 8c OS~UNDSENH. (1979) The rapid preparation of peroxisomes from rat liver by using a vertical rotor. Biochem. J. 180,445448.
OSMUNDSEN H. & BREMERJ. (1977) A spectrophotometric, procedure for rapid and sensitive measurements of /?-oxidation. Biochem. 3. 164, 621-633.
630 OSMUNDSEN H.
H. OSMUNDSEN. C. ELIZABETH NEATand B. BORREBAEK
& BREMERJ. (1978) Comparative Biochemistrv of B-oxidation. Biochem. J. 174. 3799386. OSMUNDSE~H.,‘NEATC. E. & NORUM K. R. (1979) Peroxisomal oxidation of long chain fatty acids. FEBS Mr. 99. 292-296.
OSMUNDSENH.
& NEATC. E. (1979) Regulation of peroxisomal B-oxidation. FEES Lett. 107, 81-85. SCHULTZ’H. (1974) Long chain enoyl-CoA reductase from pig heart. .I. Biol. Chem. 249, 2701-2706.