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A number of isoprenoids (e.g. pristanic acid and the side chains of fat soluble-vitamins) is degraded or shortened via beta oxidation. We synthesized 2-methyl-palmitate and 2-methyl[1-14C] palmitate as a model substrate for the study of the beta oxidation of branched (isoprenoid) fatty acids in rat liver. 2-Methylpalmitate was well oxidized by isolated hepatocytes and its oxidation was stimulated after treatment of the animals with a peroxisome proliferator. Subcellular fractionation of rat liver demonstrated that 2-methylpalmitate is activated to its CoA ester in endoplasmic reticulum, mitochondria, and peroxisomes and that mitochondria and peroxisomes are capable of beta-oxidizing 2-methylpalmitate. At low unbound 2-methylpalmitate concentrations and in the presence of competing straight chain fatty acids, a condition encountered in vivo, peroxisomal 2-methyl-palmitate oxidation was 2- to 4-fold more active than mitochondrial oxidation. Treatment of rats with a peroxisome proliferator markedly stimulated mitochondrial but only slightly peroxisomal 2-methylpalmitate oxidation. The same treatment dramatically induced palmitoyl-CoA oxidase but did not change 2-methyl-palmitoyl-CoA oxidase activity. Our results indicate 1) that in untreated rats peroxisomes contribute for an important part to the oxidation of 2-methylpalmitate; 2) that treatment with a peroxisome proliferator stimulates mainly the mitochondrial component of 2-methylpalmitate oxidation; and 3) that palmitoyl-CoA and 2-methylpalmitoyl-CoA are oxidized by different peroxisomal oxidases.

Isoprenoid (branched) fatty acids such as pristanic acid can be degraded via beta-oxidation in peroxisomes. We synthesized 2-methylpalmitoyl-CoA as a model substrate in order to study the first step of the peroxisomal beta-oxidation of branched fatty acids, catalyzed by an acyl-CoA oxidase. 2-Methylpalmitoyl-CoA oxidase activity was found in rat liver homogenates. Subcellular fractionation demonstrated that the oxidase was confined to peroxisomes. 2-Methylpalmitoyl-CoA oxidase was also present in kidney and intestine. It was not induced in liver or in the extrahepatic tissues by treatment of rats with peroxisome proliferators or by feeding diets containing excess isoprenoids. The enzyme was partially purified together with palmitoyl-CoA oxidase and trihydroxycoprostanoyl-CoA oxidase by heat treatment and ammonium sulfate fractionation of liver extracts. The partially purified preparation was chromatographed on various columns. 2-Methylpalmitoyl-CoA oxidase could be separated from the inducible (by peroxisome proliferators) palmitoyl-CoA oxidase and from trihydroxycoprostanoyl-CoA oxidase, but it always coeluted with the noninducible palmitoyl-CoA oxidase, recently described by us (Schepers, L., Van Veldhoven, P. P., Casteels, M., Eyssen, H. J., and Mannaerts, G. P. (1990) J. Biol. Chem. 265, 5242-5246). 2-Methylpalmitoyl-CoA oxidase was purified to near homogeneity in three chromatographic steps (anion exchange, hydroxylapatite, and gel filtration). Its apparent molecular mass is approximately 415 kDa, and it consists of identical subunits of approximately 70 kDa. The enzyme oxidized 2-methylpalmitoyl-CoA twice as rapidly as palmitoyl-CoA and pristanoyl-CoA as rapidly as palmitoyl-CoA, so that it can be considered as a branched fatty acyl-CoA oxidase. Since pristanoyl-CoA is one of its naturally occurring substrates we propose to name this enzyme pristanoyl-CoA oxidase.

Rat liver and kidney homogenates, fortified with the appropriate cofactors, produced H2O2 when incubated with prostaglandin (PG) E2 or its CoA ester (PGE2-CoA), indicating that PGE2-CoA served as substrate for acyl-CoA oxidase, the first enzyme of peroxisomal beta-oxidation. PGE2-CoA oxidase was stimulated to the same extent as palmitoyl-CoA oxidase by treatment of rats with the peroxisome proliferator clofibrate. Subcellular fractionation confirmed that both oxidase activities were located in peroxisomes. When highly purified peroxisomes were incubated with [1-14C]PGE2, radioactive oxidation products were released, demonstrating that peroxisomes were capable of catalyzing the complete beta-oxidation sequence. However, PGE2 beta-oxidizing activity was expressed only when isolated microsomes were added to the purified peroxisomes, indicating that PGE2-CoA synthetase is located in the endoplasmic reticulum. Cofactor requirements for peroxisomal [1-14C]PGE2 and [1-14C]palmitate oxidation were identical, and oxidation was not inhibited by cyanide. [1-14C]PGE2 was also beta-oxidized by highly purified mitochondrial fractions, devoid of peroxisomes, when microsomes were added. Mitochondrial [1-14C]PGE2 and [1-14C]palmitate oxidation was CoA- and ATP-dependent and inhibited by cyanide. Palmitate oxidation was carnitine-dependent, but PGE2 oxidation was not. Acyl-CoA dehydrogenase activity, but not carnitine acyltransferase activity, was detected with PGE2-CoA as substrate. [1-14C]PGE2 oxidation in whole liver homogenates was only slightly inhibited by cyanide, indicating that peroxisomes oxidized the major portion of PGE2. The concentrations of PGE2 employed in these experiments exceeded the in vivo concentrations by several orders of magnitude. Therefore, we suggest that the urinary PG metabolite excretion be measured in patients with hereditary diseases in which peroxisomal or mitochondrial beta-oxidation is not functioning in order to clarify the role of each organelle in PG oxidation in vivo.