Newcastle upon Tyne NE2 4HH, U.K., and IDepartment of Physiology and Biochemistry, Dental ... acid and CoA concentration on the pattern of intermediates, as.
Biochem. J. (1990) 270, 175-180 (Printed in Great Britain)
Intermediates of peroxisomal fl-oxidation A study of the fatty acyl-CoA esters which accumulate during peroxisomal fl-oxidation of
IU-"lClhexadecanoate Kim BARTLETT,* Rolf HOVIK,j Simon EATON,* Nicholas J. WATMOUGHt and Harald OSMUNDSENt§ Departments of * Child Health and t Neurology, Medical School, The University of Newcastle upon Tyne, Newcastle upon Tyne NE2 4HH, U.K., and I Department of Physiology and Biochemistry, Dental School, University of Oslo, Blindern, 0316 Oslo 3, Norway
1. 14C-labelled fatty acyl-CoA esters resulting from f-oxidation of [U-_4C]hexadecanoate by peroxisomal fractions isolated from rats treated with clofibrate showed the presence of the full range of saturated intermediates down to acetyl-CoA. 2. The pattern of intermediates generated was fairly constant. At low concentrations of [U-14C]hexadecanoate (50,M), decanoyl-CoA was present in lowest amounts. At higher -concentrations of [U-_4C]hexadecanoate (> 100 UM), all intermediates of chain length shorter than 12 carbon atoms (except acetyl-CoA) were present at similar low concentrations; the process of fl-oxidation now resembling chain-shortening of hexadecanoate by two cycles of oxidation. 3. In the absence of an NAD+-regenerating system [pyruvate and lactate dehydrogenase (EC 1.1. 1.28)] 2-enoyland 3-hydroxyacyl-CoA esters were generated, suggesting that re-oxidation of NADH is essential for optimal rates of peroxisomal fl-oxidation in vitro. 4. At high concentrations of [U-14C]hexadecanoate (> 100 /M), 3-oxohexadecanoylCoA was produced, suggesting that thiolase (acetyl-CoA acetyltransferase; EC 22.214.171.124) can become rate-limiting for peroxisomal fl-oxidation. f-
INTRODUCTION Peroxisomal fl-oxidation of long-chain fatty acids is known to be incomplete (for review, see Bremer & Osmundsen, 1984). The precise extent of chain shortening remains to be unequivocally established, although the occurrence of three to five cycles of f-oxidation has been reported for hexadecanoate (Osmundsen et al., 1979; Lazarow, 1982). It has also been reported that a fraction of the available hexadecanoate molecules may be shortened as far as butyrate (Osmundsen, 1982). Results obtained with longer (unsaturated) fatty acids suggest that two or three cycles of fl-oxidation occur (Hiltunen et al., 1986; Osmundsen & Hovik, 1988). Measurement of chain-shortening of [14C]erucic acid in vivo also suggested that two cycles of fl-oxidation is the predominant situation with respect to this substrate (Christiansen et al., 1979). Using isolated peroxisomal fractions we have examined the acyl-CoA intermediates resulting from f-oxidation of [U-14C]hexadecanoic acid. We have examined the effects of fatty acid and CoA concentration on the pattern of intermediates, as well as the effect of an added NADI-regenerating system, i.e. pyruvate plus lactate dehydrogenase (EC 126.96.36.199). Our data show that all saturated acyl-CoA esters down to acetyl-CoA are generated, although often in significantly different amounts. Depending on the conditions of incubation, 2-enoyl-, 3-hydroxyland 3-oxo-acyl-CoA esters can also be detected. EXPERIMENTAL Materials
CoA, acetyl-CoA, n-heptanoyl-CoA, n-pentadecanoyl-CoA, FAD, NADI, NADP+, Triton X-100, antimycin a, BSA (essentially fatty-acid free) and potassium pyruvate were purchased from Sigma Chemical Co., St. Louis, MO, U.S.A. All fatty acids were purchased from Fluka, Buchs, Switzerland. Percoll was obtained from Pharmacia, Uppsala, Sweden. [U-_4C]Hexadecanoic acid was purchased from Amersham International. § To whom correspondence should be addressed.
Animals Male Wistar albino rats were purchased from Veterinmr M0llegaards Avlsstasjon, Havdrup, Denmark. The rats had body weights of about 200 g at the time of experimental use. The rats were treated with clofibrate [0.5 % (w/w) in the food] for about 10 days before use. Isolation of rat liver peroxisomal fractions Rat liver peroxisomal fractions were isolated in a self-generated Percoll gradient as described previously (Neat et al., 1981; Osmundsen, 1982). The isolated fractions were used in incubations immediately after isolation. Peroxisomal incubations and assay of peroxisomal fl-oxidation Isolated peroxisomal fractions were incubated using nonsolubilizing (iso-osmotic) incubation conditions. The incubations contained (unless otherwise stated) 130 mM-KCl, 20 mM-Hepes, 0.1 mM-EGTA, 0.5 mM-NADI, 0.1 mM-NADP+, 0.2 mM-CoA, 10 mM-MgATP, 0.1 mM-dithiothreitol, 2 units of lactate dehydrogenase/ml, 20 mM-pyruvate, 10 ,tg of antimycin a/ml and 2 mg of defatted BSA/ml, pH 7.2. The substrate was always [U14C]hexadecanoate (specific radioactivity 10000 d.p.m./nmol), which was added as a complex with BSA. Unless otherwise indicated, substrate concentration was 50 /tM. The incubations contained about 0.5 mg of peroxisomal protein/ml. All incubations were carried out at 37 °C in a shaking water bath. Peroxisomal fl-oxidation of [U-14C]hexadecanoate was measured as acid-soluble radioactivity released after various periods of incubation. Usually, samples (100 ,l) of the incubation mixtures were withdrawn and added to an equivalent volume of ice-cold HC104 (10 %, v/v). Following a brief centrifugation to remove denatured proteins, the radioactivity in the supernatants was measured. The peroxisomal fractions contain a minor mitochondrial contaminant (see Neat et al., 1981). With these conditions of incubation, however, the rate of f-oxidation of labelled hexadecanoyl carnitine has been shown to be less than 10 % of that of
K. Bartlett and others
labelled hexadecanoyl-CoA (Osmundsen, 1982). When free hexadecanoate is used as a peroxisomal substrate, the resulting hexadecanoyl-CoA is essentially unavailable for mitochondrial fl-oxidation because no carnitine has been added to the incubations. It is also well established that few, if any, intermediates accumulate during mitochondrial fl-oxidation (Watmough et al., 1988, 1989). It is therefore most unlikely that a significant portion of the intermediates observed was due to contaminating mitochondrial activity. Analysis of radioactive acyl-CoA esters by radio-h.p.l.c. Incubations were quenched with glacial acetic acid and heptadecanoyl-CoA was added as internal standard. Acyl-CoA esters were extracted with methanol/chloroform (2: 1, v/v) and prepared for h.p.l.c. analysis as previously described (Watmough et al., 1989). Preliminary analysis showed that the DEAESephacel fractionation was unnecessary, and this procedure was therefore omitted. Acyl-CoA esters were analysed by h.p.l.c. with on-line radiochemical and photodiode-array detection, as described previously (Watmough et al., 1988, 1989), except that a 5 ,u Hypersil C18 column was used. Assay of protein Proteins were assayed using the Bio-Rad protein assay kit, with freeze-dried y-globulin as standard.
RESULTS Effects of pyruvate plus lactate dehydrogenase on peroxisomal p-oxidation Fig. 1 shows typical time courses of ,B-oxidation of [U200 A
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