Novel fatty acid beta-oxidation enzymes in rat liver mitochondria. II. Purification and properties of enoyl-coenzyme A (CoA) hydratase/3-hydroxyacyl-CoA dehydrogenase/3-ketoacyl-CoA thiolase trifunctional protein.

Long-chain 3-hydroxyacyl-CoA dehydrogenase was extracted from the washed membrane fraction of frozen rat liver mitochondria with buffer containing detergent and then was purified. This enzyme is an oligomer with a molecular mass of 460 kDa and consisted of 4 mol of large polypeptide (79 kDa) and 4 mol of small polypeptides (51 and 49 kDa). The purified enzyme preparation was concluded to be free from the following enzymes based on marked differences in behavior of the enzyme during purification, molecular masses of the native enzyme and subunits, and immunochemical properties: enoyl-CoA hydratase, short-chain 3-hydroxyacyl-CoA dehydrogenase, peroxisomal enoyl-CoA hydratase/3-hydroxyacyl-CoA dehydrogenase bifunctional protein, and mitochondrial and peroxisomal 3-ketoacyl-CoA thiolases. The purified enzyme exhibited activities toward enoyl-CoA hydratase and 3-ketoacyl-CoA thiolase together with the long-chain 3-hydroxyacyl-CoA dehydrogenase activity. The carbon chain length specificities of these three activities of this enzyme differed from those of the other enzymes. Therefore, it is concluded that this enzyme is not long-chain 3-hydroxyacyl-CoA dehydrogenase; rather, it is enoyl-CoA hydratase/3-hydroxyacyl-CoA dehydrogenase/3-ketoacyl-CoA thiolase trifunctional protein.     

Changes in the activities of the enzymes of hepatic fatty acid oxidation during development of the rat.

1. Changes in the activities of several enzymes involved in mitochondrial fatty acid oxidation were measured in livers of developing rats between late foetal life and maturity. The enzymes studied are medium- and long-chain ATP-dependent acyl-CoA synthetases of the outer mitochondrial membrane and matrix, GTP-dependent acyl-CoA synthetase, carnitine acyltransferase, enoyl-CoA hydratase, 3-hydroxyacyl-CoA dehydrogenase, general 3-oxoacyl-CoA thiolase and acetoacetyl-CoA thiolase.     

Induction of acyl coenzyme A synthetase and hydroxyacyl coenzyme A dehydrogenase during fatty acid degradation in Neurospora crassa

Neurospora crassa is able to use long-chain fatty acids as the sole carbon and energy source. After growth on oleate there was nearly a 10-fold induction of the acyl coenzyme A (CoA) synthetase and a fivefold increase in the activity of the 3-hydroxyacyl-CoA dehydrogenase. There was a slight induction of the enoyl-CoA hydratase and 3-ketoacyl-CoA thiolase, but no apparent induction of the flavin-linked acyl-CoA dehydrogenase. These noncoordinate changes in the fatty acid degradation enzymes suggest that they are not organized into a multienzyme complex as is found in bacteria.     

Human liver long-chain 3-hydroxyacyl-coenzyme A dehydrogenase is a multifunctional membrane-bound beta-oxidation enzyme of mitochondria.

We have purified to homogeneity the long-chain specific 3-hydroxyacyl-CoA dehydrogenase from mitochondrial membranes of human infant liver. The enzyme is composed of non-identical subunits of 71 kDa and 47 kDa within a native structure of 230 kDa. The pure enzyme is active with 3-ketohexanoyl-CoA and gives maximum activity with 3-ketoacyl-CoA substrates of C10 to C16 acyl-chain length but is inactive with acetoacetyl-CoA. In addition to 3-hydroxyacyl-CoA dehydrogenase activity, the enzyme possesses 2-enoyl-CoA hydratase and 3-ketoacyl-CoA thiolase activities which cannot be separated from the dehydrogenase. None of these enzymes show activity with C4 substrates but all are active with C6 and longer acyl-chain length substrates. They are thus distinct from any described previously. This human liver mitochondrial membrane-bound enzyme catalyses the conversion of medium- and long-chain 2-enoyl-CoA compounds to: 1) 3-ketoacyl-CoA in the presence of NAD alone and 2) to acetyl-CoA (plus the corresponding acyl-CoA derivatives) in the presence of NAD and CoASH. It is therefore a multifunctional enzyme, resembling the beta-oxidation enzyme of E. coli, but unique in its membrane location and substrate specificity. We propose that its existence explains the repeated failure to detect any intermediates of mitochondrial beta-oxidation.     

Identification of enzymes involved in oxidation of phenylbutyrate

In recent years the short-chain fatty acid, 4-phenylbutyrate (PB), has emerged as a promising drug for various clinical conditions. In fact, PB has been Food and Drug Administration-approved for urea cycle disorders since 1996. PB is more potent and less toxic than its metabolite, phenylacetate (PA), and is not just a pro-drug for PA, as was initially assumed. The metabolic pathway of PB, however, has remained unclear. Therefore, we set out to identify the enzymes involved in the β-oxidation of PB. We used cells deficient in specific steps of fatty acid β-oxidation and ultra-HPLC to measure which enzymes were able to convert PB or its downstream products. We show that the first step in PB oxidation is catalyzed solely by the enzyme, medium-chain acyl-CoA dehydrogenase. The second (hydration) step can be catalyzed by all three mitochondrial enoyl-CoA hydratase enzymes, i.e., short-chain enoyl-CoA hydratase, long-chain enoyl-CoA hydratase, and 3-methylglutaconyl-CoA hydratase. Enzymes involved in the third step include both short- and long-chain 3-hydroxyacyl-CoA dehydrogenase. The oxidation of PB is completed by only one enzyme, i.e., long-chain 3-ketoacyl-CoA thiolase. Taken together, the enzymatic characteristics of the PB degradative pathway may lead to better dose finding and limiting the toxicity of this drug.     

Peroxisomal Localization of Enzymes Related to Fatty Acid β-Oxidation in an n-Alkane-grown Yeast,Candida tropicalis

In Candida tropicalis cells grown on n-alkanes (C₁₀-C₁₃), the levels of the activities of the enzymes related to fatty acid β—oxidation—acyl-CoA oxidase, enoyl-CoA hydratase, 3-hydroxyacyl-CoA dehydrogenase and 3-ketoacyl-CoA thiolase—were found to be higher than those in cells grown on glucose, indicating that these enzymes were induced by alkanes. The enzymes were first confirmed to be localized only in peroxisomes, while none of these enzymes nor acyl-CoA dehydrogenase, which is known to participate in the initial step of mitochondrial β-oxidation in mammalian cells, were detected in yeast mitochondria under the conditions employed.

The significance of the peroxisomal β-oxidation system in the metabolism of alkanes by the yeast was also discussed.     

Mitochondrial metabolism of valproic acid

The beta-oxidation of valproic acid (2-propylpentanoic acid), an anticonvulsant drug with hepatotoxic side effects, was studied with subcellular fractions of rat liver and with purified enzymes of beta-oxidation. 2-Propyl-2-pentenoyl-CoA, a presumed intermediate in the beta-oxidation of valproic acid, was chemically synthesized and used to demonstrate that enoyl-CoA hydratase or crotonase catalyzes its hydration to 3-hydroxy-2-propylpentanoyl-CoA. The latter compound was not acted upon by soluble L-3-hydroxyacyl-CoA dehydrogenases from mitochondria or peroxisomes but was dehydrogenated by an NAD(+)-dependent dehydrogenase associated with a mitochondrial membrane fraction. The product of the dehydrogenation, presumably 3-keto-2-propylpentanoyl-CoA, was further characterized by fast bombardment mass spectrometry. 3-Keto-2-propylpentanoyl-CoA was not cleaved thiolytically by 3-ketoacyl-CoA thiolase or a mitochondrial extract but was slowly degraded, most likely by hydrolysis. The availability of 2-propylpentanoyl-CoA (valproyl-CoA) and its beta-oxidation metabolites facilitated a study of valproate metabolism in coupled rat liver mitochondria. Mitochondrial metabolites identified by high-performance liquid chromatography were 2-propylpentanoyl-CoA, 3-keto-2-propylpentanoyl-CoA, 2-propyl-2-pentenoyl- CoA, and trace amounts of 3-hydroxy-2-propylpentanoyl-CoA. It is concluded that valproic acid enters mitochondria where it is converted to 2-propylpentanoyl-CoA, dehydrogenated to 2-propyl-2-pentenoyl-CoA by 2-methyl-branched chain acyl-CoA dehydrogenase, and hydrated by enoyl-CoA hydratase to 3-hydroxy-2-propylpentanoyl-CoA.(ABSTRACT TRUNCATED AT 250 WORDS)     

Biochemical effects of the hypoglycaemic compound pent-4-enoic acid and related non-hypoglycaemic fatty acids. Effects of their coenzyme A esters on enzymes of fatty acid oxidation

1. Pent-4-enoyl-CoA and its metabolites penta-2,4-dienoyl-CoA and acryloyl-CoA, as well as n-pentanoyl-CoA, cyclopropanecarbonyl-CoA and cyclobutanecarbonyl-CoA, were examined as substrates or inhibitors of purified enzymes of beta-oxidation in an investigation to locate the site of inhibition of fatty acid oxidation by pent-4-enoate. 2. The reactions of various acyl-CoA derivatives with l-carnitine and of various acyl-l-carnitine derivatives with CoA, catalysed by carnitine acetyltransferase, were investigated and V(max.) and K(m) values were determined. Pent-4-enoyl-CoA and n-pentanoyl-CoA were good substrates, whereas cyclobutanecarbonyl-CoA, cyclopropanecarbonyl-CoA and acryloyl-CoA reacted more slowly. A very slow rate with penta-2,4-dienoyl-CoA was detected. Pent-4-enoyl-l-carnitine, n-pentanoyl-l-carnitine and cyclobutanecarbonyl-l-carnitine were good substrates and cyclopropanecarbonyl-l-carnitine reacted more slowly. 3. Pent-4-enoyl-CoA and n-pentanoyl-CoA were substrates for butyryl-CoA dehydrogenase and for octanoyl-CoA dehydrogenase, and both compounds were equally effective competitive inhibitors of these enzymes with butyryl-CoA or palmitoyl-CoA respectively as substrates. V(max.), K(m) and K(i) values were determined. 4. None of the acyl-CoA derivatives inhibited enoyl-CoA hydratase or 3-hydroxybutyryl-CoA dehydrogenase. Penta-2,4-dienoyl-CoA was a substrate for enoyl-CoA hydratase when the reaction was coupled to that catalysed by 3-hydroxybutyryl-CoA dehydrogenase. 5. In a reconstituted sequence with purified enzymes crotonoyl-CoA was largely converted into acetyl-CoA, and pent-2-enoyl-CoA into acetyl-CoA and propionyl-CoA. Penta-2,4-dienoyl-CoA was slowly converted into acetyl-CoA and acryloyl-CoA. 6. Penta-2,4-dienoyl-CoA, a unique metabolite of pent-4-enoate, was the only compound that specifically inhibited an enzyme of the beta-oxidation sequence, 3-oxoacyl-CoA thiolase. The formation of penta-2,4-dienoyl-CoA could explain the strong inhibition of fatty acid oxidation in intact mitochondria by pent-4-enoate.     

Transport of proteins into mitochondrial matrix. Evidence suggesting a common pathway for 3-ketoacyl-CoA thiolase and enzymes having presequences

Rat liver 3-ketoacyl-CoA thiolase, a mitochondrial matrix enzyme which catalyzes a step of fatty acid beta-oxidation, was synthesized in a rabbit reticulocyte lysate cell-free system. The in vitro product was apparently the same in molecular size and charge as the subunit of the mature enzyme. The enzyme synthesized in vitro was transported into isolated rat liver mitochondria in an energy-dependent manner. In pulse experiments with isolated rat hepatocytes at 37 degrees C, the radioactivity of the newly synthesized enzyme in the cytosolic fraction remained essentially unchanged during 5-20 min of incubation, whereas that of the enzyme in the particulate fraction increased with time during the incubation. The pulse-labeled enzyme disappeared with an apparent half-life of less than 3 min from the cytosolic fraction, in pulse-chase experiments. Purified 3-ketoacyl-CoA thiolase inhibited the mitochondrial uptake and processing of the precursors of the other matrix enzymes, ornithine carbamoyltransferase, medium-chain acyl-CoA dehydrogenase and acetoacetyl-CoA thiolase. These results indicate that 3-ketoacyl-CoA thiolase has an internal signal which is recognized by the mitochondria and suggest that this enzyme and the three others are transported into the mitochondria by a common pathway.     

Peroxisomal beta-oxidation enzyme proteins in the Zellweger syndrome

The absence of peroxisomes in patients with the cerebro-hepato-renal (Zellweger) syndrome is accompanied by a number of biochemical abnormalities, including an accumulation of very long-chain fatty acids. We show by immunoblotting that there is a marked deficiency in livers from patients with the Zellweger syndrome of the peroxisomal beta-oxidation enzyme proteins acyl-CoA oxidase, the bifunctional protein with enoyl-CoA hydratase and 3-hydroxyacyl-CoA dehydrogenase activities and 3-oxoacyl-CoA thiolase. Using anti-(acyl-CoA oxidase), increased amounts of cross-reactive material of low Mr were seen in the patients. With anti-(oxoacyl-CoA thiolase), high Mr cross-reactive material, presumably representing precursor forms of 3-oxoacyl-CoA thiolase, was detected in the patients. Catalase protein was not deficient, in accordance with the finding that catalase activity is not diminished in the patients. Thus in contrast to the situation with catalase functional peroxisomes are required for the stability and normal activity of peroxisomal beta-oxidation enzymes.     

Acetyl-CoA-dependent elongation of fatty acids in Mycobacterium smegmatis.

An enzyme system of Mycobacterium smegmatis catalyzing the elongation of medium-chain fatty acids with acetyl-CoA was obtained free from de novo fatty acid synthetase by ammonium sulfate fractionation. The system was resolved by gel filtration and DEAE-cellulose chromatography into three fractions, all of which were required for reconstitution of the elongation activity. The three fractions were highly purified enoyl-CoA hydratase, highly purified 3-hydroxyacyl-CoA dehydrogenase, and a fraction containing both enoyl-CoA reductase and thiolase. The reconstituted system was avidin-insenstive, required NADH as a sole hydrogen donor, and was sensitive to pCMB, but not to N-ethylmaleimide or monoiodoacetate. Decanoyl-CoA and octanoyl-CoA were the best primers for the elongation system. When decanoyl-CoA was used as the primer, the major product was found to be a lauroyl derivative (probably lauroyl-CoA). Evidence was obtained suggesting that acyl-CoA dehydrogenase, catalyzing the first step of beta-oxidation, was not functional in the elongation system.     

Mitochondrial enzymes responsible for oxidizing medium-chain fatty acids in developing rat skeletal muscle, heart, and liver

Prior to weaning, medium-chain fatty acids constitute an important energy source in the developing rat. Fatty acid oxidation rates increase with age in most developing tissues, but the pattern of this increase may vary according to the role of the particular organ. In skeletal muscle, heart, and liver of developing rats, we measured mitochondrial activities of long- and short-chain enoyl-CoA hydratase, 3-hydroxyacyl-CoA dehydrogenase, and long- and short-chain acyl-CoA thiolase. In skeletal muscle, the pattern of development in fatty acid oxidation enzymes favored utilization of long-chain rather than medium-chain fatty acids. In liver, enzyme activities for medium-chain fatty acids were highest prior to weaning. Heart occupied a position intermediate between skeletal muscle and liver.     

Enzymes of beta-Oxidation in Different Types of Algal Microbodies

The algae Mougeotia and Eremosphaera were used for isolation of microbodies with the characteristics of leaf peroxisomes and unspecialized peroxisomes, respectively. In both types of organelles, the following enzymes of the β-oxidation pathway were determined: acyl-CoA oxido-reductase, enoyl-CoA hydratase, and 3-hydroxyacyl-CoA dehydrogenase. There are indications that the peroxisomal oxidoreductase of both algae is a H₂O₂-forming oxidase rather than a dehydrogenase.

The enzymes enoyl-CoA hydratase and acyl-CoA oxidoreductase are located also in the mitochondria from Eremosphaera but not from Mougeotia. The mitochondrial acyl-CoA oxidizing enzyme was found to be a dehydrogenase. The specific activities of acyl-CoA oxidase and enoyl-CoA hydratase are lower than in spinach leaf peroxisomes. However, the activity of 3-hydroxyacyl-CoA dehydrogenase in the peroxisomes of both algae is almost 2-fold higher. The capability for degradation of fatty acids is a common feature of all different types of peroxisomes from algae.

     

Evidence that the fadB gene of the fadAB operon of Escherichia coli encodes 3-hydroxyacyl-coenzyme A (CoA) epimerase, delta 3-cis-delta 2-trans-enoyl-CoA isomerase, and enoyl-CoA hydratase in addition to 3-hydroxyacyl-CoA dehydrogenase.

Genetic complementation of a mutant defective in fatty acid oxidation (fadAB) with plasmids containing DNA inserts from the fadAB region of the Escherichia coli genome was studied. The mutant containing the hybrid plasmid with a 5.2-kilobase (kb) PstI-SalI fragment was found to overproduce 3-hydroxyacyl-coenzyme A (CoA) epimerase and delta 3-cis-delta 2-trans-enoyl-CoA isomerase as well as three other beta-oxidation enzymes by 16- to 18-fold compared with the wild-type parental strain LE392. The purification of a fully functional multienzyme complex of fatty acid oxidation from the transformant ultimately established that the 5.2-kb DNA fragment contained an entire fadAB operon. Since immunotitration of cell extracts with antibodies against the fatty acid oxidation complex proved that all 3-hydroxyacyl-CoA epimerase and delta 3-cis-delta 2-trans-enoyl-CoA isomerase activities were associated with the complex, no genetic loci other than the fadAB operon encoded these two enzymes. Moreover, the binding of antibodies caused parallel inhibition of four component enzymes, whereas 3-ketoacyl-CoA thiolase activity was slightly increased. These findings support the suggestion that the epimerase and isomerase as well as enoyl-CoA hydratase and L-3-hydroxyacyl-CoA dehydrogenase are located on the same polypeptide. The results of this study, together with published data (S.-Y. Yang and H. Schulz, J. Biol. Chem. 258:9780-9785, 1983), lead to the conclusion that 3-hydroxyacyl-CoA epimerase, delta 3-cis-delta 2-trans-enoyl-CoA isomerase, and enoyl-CoA hydratase in addition to 3-hydroxyacyl-CoA dehydrogenase are encoded by the fadB gene.     

The mitochondrial trifunctional protein: centre of a beta-oxidation metabolon?

The trifunctional enzyme comprises three consecutive steps in the mitochondrial beta-oxidation of long-chain acyl-CoA esters: 2-enoyl-CoA hydratase, 3-hydroxyacyl-CoA dehydrogenase and 3-ketoacyl-CoA thiolase. Deficiencies in either 3-hydroxyacyl-CoA dehydrogenase activity, or all three activities, are important causes of human disease. The dehydrogenase and thiolase have a requirement for NAD+ and CoA respectively, whose levels are conserved within the mitochondrion and thus provide possible means for control and regulation of beta-oxidation. Using analysis of the intact CoA ester intermediates produced by the complex, we have examined the sensitivity of the complex to NAD+/NADH and acetyl-CoA. We consider the evidence for channelling within the trifunctional protein and propose a model for a beta-oxidation 'metabolon'.     

Significance of catalase in peroxisomal fatty acyl-CoA beta-oxidation

Catalase activity was inhibited by aminotriazole administration to rats in order to evaluate the influence of catalase on the peroxisomal fatty acyl-CoA beta-oxidation system. 2 h after the administration of aminotriazole, peroxisomes were prepared from rat liver, and the activities of catalase, the beta-oxidation system and individual enzymes of beta-oxidation (fatty acyl-CoA oxidase, crotonase, beta-hydroxybutyryl-CoA dehydrogenase and thiolase) were determined. Catalase activity was decreased to about 2% of the control. Among the individual enzymes of the beta-oxidation system, thiolase activity was decreased to 67%, but the activities of fatty acyl-CoA oxidase, crotonase and beta-hydroxybutyryl-CoA dehydrogenase were almost unchanged. The activity of the peroxisomal beta-oxidation system was assayed by measuring palmitoyl-CoA-dependent NADH formation, and the activity of the purified peroxisome preparation was found to be almost unaffected by the administration of aminotriazole. The activity of the system in the aminotriazole-treated preparation was, however, significantly decreased to 55% by addition of 0.1 mM H2O2 to the incubation mixture. Hydrogen peroxide (0.1 mM) reduced the thiolase activity of the aminotriazole-treated peroxisomes to approx. 40%, but did not affect the other activities of the system. Thiolase activity of the control preparation was decreased to 70% by addition of hydrogen peroxide (0.1 mM). The half-life of 0.1 mM H2O2 added to the thiolase assay mixture was 2.8 min in the case of aminotriazole-treated peroxisomes, and 4 s in control peroxisomes. The ultraviolet spectrum of acetoacetyl-CoA (substrate of thiolase) was clearly changed by addition of 0.1 mM H2O2 to the thiolase assay mixture without the enzyme preparation; the absorption bands at around 233 nm (possibly due to the thioester bond of acetoacetyl-CoA) and at around 303 nm (due to formation of the enolate ion) were both significantly decreased. These results suggest that H2O2 accumulated in peroxisomes after aminotriazole treatment may modify both thiolase and its substrate, and consequently suppress the fatty acyl-CoA beta-oxidation. Therefore, catalase may protect thiolase and its substrate, 3-ketoacyl-CoA, by removing H2O2, which is abundantly produced during peroxisomal enzyme reactions.     

Intracellular Localization of Enzymes of Fatty Acid-beta-Oxidation in the Alga Cyanidium caldarium

The intracellular distribution of enzymes, participating in the β-oxidation of fatty acids in the eucaryotic alga Cyanidium has been studied. After separating the organelles from a crude homogenate on a linear flotation gradient, the enzymes enoyl-CoA hydratase, hydroxyacyl-CoA dehydrogenase, and thiolase were present in the mitochondrial fraction (density: 1.19 gram per cubic centimeter). Activity of an acyl-CoA synthetase was found in the mitochondrial fraction as well as in a band where mitochondrial membrane apparently had accumulated (density: 1.17 gram per cubic centimeter). None of these enzymes were present in the peroxisomes (density: 1.23 gram per cubic centimeter). Results from cell fractionation as well as properties of β-oxidation enzymes indicate a mitochondrial location of fatty acid degradation also in the algae Galdieria sulphuraria and Cyanidioschyzon merolae.     

The effect of treatment of rats with pituitary growth hormone on the activities of some enzymes involved in fatty acid degradation and synthesis

1. Measurements have been made of the activities of acyl-CoA dehydrogenase, enoyl-CoA hydratase, beta-hydroxyacyl-CoA dehydrogenase and ketothiolase in the livers of rats treated for either 12hr. or 3 days with pituitary growth hormone. 2. There was a significant increase in the activity of acyl-CoA dehydrogenase in rats treated with the hormone for 3 days. 3. Measurements were also made of the lipogenic enzymes acetyl-CoA carboxylase and palmitate synthase in the livers of similarly treated animals. 4. There was a depression of the activity of both enzymes after 12hr. treatment and a further decline after 3 days. 5. The results are discussed in relation to the known increase in the rate of fatty acid oxidation and inhibition of fatty acid synthesis in rats treated with growth hormone.     

Fatty acid oxidation in rat brain is limited by the low activity of 3-ketoacyl-coenzyme A thiolase

In an attempt to clarify why the brain oxidizes fatty acids poorly or not at all, the activities of beta-oxidation enzymes present in rat brain and rat heart mitochondria were measured and compared with each other. Although the apparent Km values and chain-length specificities of the brain and heart enzymes are similar, the specific activities of all but one brain enzyme are between 4 and 50% of those observed in heart mitochondria. The exception is 3-ketoacyl-CoA thiolase (EC 2.3.1.16) whose specific activity in brain mitochondria is 125 times lower than in heart mitochondria. The partially purified brain 3-ketoacyl-CoA thiolase was shown to be catalytically and immunologically identical with the heart enzyme. The low rate of fatty acid oxidation in brain mitochondria, estimated on the basis of palmitoylcarnitine-supported respiration and [1-14C]palmitoylcarnitine degradation to be less than 0.5 nmol/min/mg of protein, may be the consequence of the low activity of 3-ketoacyl-CoA thiolase. Inhibition of [1-14C]palmitoylcarnitine oxidation by 4-bromocrotonic acid proves the observed oxidation of fatty acids in brain to be dependent on 3-ketoacyl-CoA thiolase and thus to occur via beta-oxidation. Since the reactions catalyzed by carnitine palmitoyltransferase (EC 2.3.1.21) and acyl-CoA synthetase (EC 6.2.1.3) do not seem to restrict fatty acid oxidation in brain, it is concluded that the oxidation of fatty acids in rat brain is limited by the activity of the mitochondrial 3-keto-acyl-CoA thiolase.     

Innermembrane association of three mitochondrial beta-oxidation enzymes revealed by immunoelectron microscopic technique

Ultrastructural localization of three mitochondrial beta-oxidation enzymes, enoyl-CoA hydratase, 3-hydroxyacyl-CoA dehydrogenase, and 3-ketoacyl-CoA thiolase in rat liver was studied by a post-embedding immunocytochemical technique. Rat liver was fixed by perfusion. Vibratome sections (100 micron thick) were embedded in Lowicryl K4M. Ultrathin sections were separately incubated with antibody to each enzyme, followed by protein A-gold complex. Gold particles representing the antigenic sites for all enzymes examined were confined exclusively to mitochondria of hepatocytes and other sinus-lining cells. Peroxisomes were consistently negative for the immunolabelling. In the mitochondria the gold particles were localized in the matrical side of inner membrane. The control experiments confirmed the specificity of the immunolabelling. The results firstly indicate that the mitochondrial beta-oxidation enzymes are present in the matrix of mitochondria and associated with the inner membrane.