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(1) During peroxisomal beta-oxidation of [U-14C]hexadecanoate, at concentrations higher than 100 microM, long-chain 3-oxoacyl-CoA-esters and 3-oxobutyryl-CoA accumulate. Only 3-oxobutyryl-CoA accumulates at a low concentration of [U-14C]hexadecanoate. Accumulation of long chain 3-oxoacyl-CoA esters is most extensive when the supply of CoA can be considered limiting for beta-oxidation. (2) Added acetyl-CoA was found to inhibit peroxisomal beta-oxidation. This inhibition was not significantly relieved by added L-carnitine and carnitine acetyltransferase (EC 2.3.17). (3) Added L-carnitine, at concentrations below 0.2 mM, was found to stimulate peroxisomal beta-oxidation of [U-14C]hexadecanoate by up to 20%, causing the conversion of acetyl-CoA into acetylcarnitine. Higher concentrations of L-carnitine were progressively inhibitory to beta-oxidation. This effect was specific for L-carnitine as both D-carnitine and aminocarnitine neither caused stimulation at low concentrations, nor inhibition at higher concentrations. Added L-carnitine caused accumulation of acylcarnitines of chain-lengths ranging from 4 to 16 carbon-atoms. The inhibition observed with higher concentrations of added L-carnitine is likely due to conversion of [U-14C]hexadecanoate into [U-14C]hexadecanoylcarnitine. (4) Low concentrations of added hexadecanoylcarnitine was shown to inhibit peroxisomal beta-oxidation by about 15%, while added acetylcarnitine did not inhibit at concentrations up to 100 microM. (5) These data are interpreted to indicate significant control being exerted on flux at the stage of thiolysis either directly by means of CoA availability, or indirectly by means of the rate of acetyl-CoA generation.

The activities of acyl-CoA hydrolase (EC 3.1.2.2.) and acyl-CoA:1-acyl- lysophospholipid acyltransferase (EC 2.3.1.23) have been studied in subcellular fractions of human platelets. The acyl-CoA:1-acyl-lysophospholipid acyltransferase activity was higher in the 'dense-tubular-system-enriched' fraction than in the 'light-mitochondrial' fraction, using endogenously acyl-CoAs formed from labelled fatty acids, ATP, CoA and various lysophospholipids. No activity was found in the 'particle-free' fraction. No difference in specificities was observed between the incorporation of various fatty acids into different lysoPLs in the subcellular fractions compared with the platelet lysates. Generally, arachidonic, linoleic and eicosapentaenoic acids were better substrates for the acyl-CoA:1-acyl-lysophospholipid acyltransferases than oleic, docosahexaenoic and palmitic acids. The opposite was observed with the acyl-CoA hydrolase activity, palmitoyl-CoA was the substrate giving the highest activity, and eicosapentaenoyl-CoA and arachidonoyl-CoA the lowest. About 85% of the hydrolase activity was detected in the 'particle-free' fraction, with each of the six acyl-CoA derivatives tested.

In the course of the last decade peroxisomal beta-oxidation has emerged as a metabolic process indispensable to normal physiology. Peroxisomes beta-oxidize fatty acids, dicarboxylic acids, prostaglandins and various fatty acid analogues. Other compounds possessing an alkyl-group of six to eight carbon atoms (many substituted fatty acids) are initially omega-oxidized in endoplasmic reticulum. The resulting carboxyalkyl-groups are subsequently chain-shortened by beta-oxidation in peroxisomes. Peroxisomal beta-oxidation is therefore, in contrast to mitochondrial beta-oxidation, characterized by a very broad substrate-specificity. Acyl-CoA oxidases initiate the cycle of beta-oxidation of acyl-CoA esters. The next steps involve the bi(tri)functional enzyme, which possesses active sites for enoyl-CoA hydratase-, beta-hydroxyacyl-CoA dehydrogenase- and for delta 2, delta 5 enoyl-CoA isomerase activity. The beta-oxidation sequence is completed by a beta-ketoacyl-CoA thiolase. The peroxisomes also contain a 2,4-dienoyl-CoA reductase, which is required for beta-oxidation of unsaturated fatty acids. The peroxisomal beta-hydroxyacyl-CoA epimerase activity is due to the combined action of two enoyl-CoA hydratases. (For a recent review of the enzymology of beta-oxidation enzymes see Ref. 225.) The broad specificity of peroxisomal beta-oxidation is in part due to the presence of at least two acyl-CoA oxidases, one of which, the trihydroxy-5 beta-cholestanoyl-CoA (THCA-CoA) oxidase, is responsible for the initial dehydrogenation of the omega-oxidized cholesterol side-chain, initially hydroxylated in mitochondria. Shortening of this side-chain results in formation of bile acids and of propionyl-CoA. In relation to its mitochondrial counterpart, peroxisomal beta-oxidation in rat liver is characterized by a high extent of induction following exposure of rats to a variety of amphipathic compounds possessing a carboxylic-, or sulphonic acid group. In rats some high fat diets cause induction of peroxisomal fatty acid beta-oxidation and of trihydroxy-5 beta-cholestanoyl-CoA oxidase. Induction involves increased rates of synthesis of the appropriate mRNA molecules. Increased half-lives of mRNA- and enzyme molecules may also be involved. Recent findings of the involvement of a member of the steroid hormone receptor superfamily during induction, suggest that induction of peroxisomal beta-oxidation represents another regulatory phenomenon controlled by nuclear receptor proteins. This will likely be an area of intense future research. Chain-shortening of fatty acids, rather than their complete beta-oxidation, is the prominent feature of peroxisomal beta-oxidation.(ABSTRACT TRUNCATED AT 400 WORDS)

beta-Oxidation of polyunsaturated fatty acids was studied with isolated rat liver mitochondria in state 3 or uncoupled conditions. 1. Incubation of mitochondria with docosahexaenoyl-, linolenoyl- or gamma-linolenoylcarnitine resulted in an increase of the absorbance at 340 minus 385 nm. This increased absorbance was due to an accumulation of beta-oxidation intermediates of the polyunsaturated fatty acids, and not to the reduction of nicotinamide nucleotides. 2. Experiments carried out with soluble fractions of liver mitochondria incubated with docosahexaenoyl-CoA and gamma-linolenoyl-CoA indicated that this ultraviolet light-absorption was at least partly caused by acyl-CoA esters having a 2,4(,7)-di(tri)enoyl-CoA structure. 3. The addition of glutamate to mitochondria oxidizing gamma-linolenoylcarnitine decreased the absorbance at 340 minus 385 nm, and simultaneously stimulated respiration. With liver mitochondria isolated from fasted rats, 6 mM glutamate increased the rate of acetoacetate production from gamma-linolenoylcarnitine by 130 and 210% under state 3 and uncoupled conditions, respectively. Glutamate did not have any significant effect on the degradation of oleoylcarnitine. The proposed explanation for these findings is that the glutamate dehydrogenase reaction can function as a source of NADPH for 2,4-dienoyl-CoA reductase. 4. The degradation of gamma-linolenoylcarnitine to ketone bodies was augmented in mitochondria isolated from rats treated with clofibrate or partially hydrogenated marine oil. 5. We conclude that 2,4-dienoyl-CoA reductase is an important auxiliary enzyme in the beta-oxidation of polyunsaturated fatty acids. Induction of this enzyme by clofibrate or by certain high-fat diets increases mitochondrial capacity for the degradation of polyunsaturated fatty acids.

Among subcellular fractions of liver homogenates of rats, the clofibroyl-CoA hydrolase activity is found mainly in the cytosolic fraction. It is here shown that the subcellular distribution of clofibroyl-CoA hydrolase appears to be different from the distribution of palmitoyl-CoA hydrolase activity. Thus, in contrast to the case with palmitoyl-CoA, no hydrolysis of clofibroyl-CoA was catalysed by the microsomal fraction. Furthermore, the hydrolysis of palmitoyl-CoA and clofibroyl-CoA in the cytosolic fraction seemed to be catalyzed by two different enzymes. Rats treated with clofibrate (0.3%, w/w) showed a significant increased clofibroyl-CoA hydrolase activity where the cytosolic hydrolase was increased 3.5-fold. Clofibrate administration also elevated the specific clofibroyl-CoA hydrolase activity by factors of 1.7 and 1.5 in the mitochondrial and the light-mitochondrial fractions, respectively. Thus, it is possible that clofibroyl-CoA hydrolase has also a multiorganelle localization.

Butyryl-coenzyme A synthetase (butyrate:CoA ligase (AMP-forming), EC 6.2.1.2) from an acetone-dried powder of ox liver mitochondria was found to have a molecular weight of approx. 40 000. The sedimentation equilibrium analysis suggested the presence in solution of higher molecular weight forms of the enzyme and these could also be obtained by extracting the enzyme from the mitochondrial powder in non-reducing conditions. The enzyme was inhibited by sulphydryl reagents, and was found to have at least one available thiol group/molecule. The relationship between enzymic activity and concentration was non-linear, and suggested that an inactive monomer-active dimer equilibrium was present. The 5--6-fold activation by bovine serum albumin required the presence of free thiol groups in the albumin and involved association of albumin with the enzyme.

Pyrenedodecanoyl-CoA was beta-oxidized by isolated rat liver peroxisomes at a rate which was about 50% of that observed with palmitoyl-CoA. Measurement of the quantity of NADH formed from a limiting amount of pyrenedodecanoyl-CoA suggested that it was subjected to two to three cycles of beta-oxidation. Pyrenedodecanoyl-CoA was a very poor substrate for carnitine palmitoyltransferase, exhibiting less than 1% of the rate obtained with palmitoyl-CoA; it also was a strong inhibitor of this enzyme. With rat liver microsomal alpha-glycerophosphate acyltransferase the rate of reaction with pyrenedodecanoyl-CoA was only 3-4% of that observed with palmitoyl-CoA.