Comparison of the stability and substrate specificity of purified peroxisomal 3-oxoacyl-CoA thiolases A and B from rat liver

The specific activities and substrate specificities of 3-oxoacyl-CoA thiolase A (thiolase A) purified from normal rat liver peroxisomes and 3-oxoacyl-CoA thiolase B (thiolase B) isolated from livers of rats treated with the peroxisome proliferator clofibrate were virtually identical. The enzymes could be distinguished by their N-terminal amino acid sequences, their isoelectric points and their stability, the latter being higher for thiolase A. Contrary to thiolase B, which showed a marked cold lability in the presence of KCl by dissociating into monomers with poor activity, thiolase A retained its full activity and its homodimeric structure under these conditions.     

Molecular cloning of cDNA for rat mitochondrial 3-oxoacyl-CoA thiolase

Messenger RNA of rat 3-oxoacyl-CoA thiolase (acetyl-CoA acyltransferase), a mitochondrial matrix enzyme involved in fatty acid beta-oxidation, was enriched by immunoprecipitation of rat liver free polysomes and recombinant plasmids were prepared from the enriched mRNA by a modification of the vector-primer method of Okayama and Berg. The transformants were initially screened for 3-oxoacyl-CoA thiolase cDNA sequences by differential colony hybridization with [32P]cDNAs, synthesized from the immunopurified and unpurified mRNAs. The cDNA clones for 3-oxoacyl-CoA thiolase were identified by hybrid-arrested translation and hybrid-selected translation. One of the clones, designated pT1-1, contained a 700-base insert and hybridized to a mRNA species of 1.6 X 10(3) bases in rat liver. The transformants were rescreened using the cDNA insert of pT1-1 as a hybridization probe and a clone (pT1-19) with a 1.5 X 10(3)-base insert was obtained. Activity and concentration of 3-oxoacyl-CoA thiolase mRNA were quantified by in vitro translation and dot-blot analysis using the cDNA insert as a hybridization probe. The level of translatable and hybridizable mRNA in rat liver was increased about 5.1-fold and 4.6-fold, respectively, after administration of di-(2-ethylhexyl)phthalate, a potent inducer of the enzyme. The 3-oxoacyl-CoA thiolase mRNA levels thus determined correlated closely with levels of the activity and amount of this enzyme.     

cDNA-derived amino acid sequence of rat mitochondrial 3-oxoacyl-CoA thiolase with no transient presequence: structural relationship with peroxisomal isozyme.

The sorting of homologous proteins between two separate intracellular organelles is a major unsolved problem. 3-Oxoacyl-CoA thiolase is localized in mitochondria and peroxisomes, and provides a good system for the study on the problem. Unlike most mitochondrial matrix proteins, mitochondrial 3-oxoacyl-CoA thiolase in rats is synthesized with no transient presequence and possess information for mitochondrial targeting and import in the mature protein. Two overlapping cDNA clones contained an open reading frame encoding a polypeptide of 397 amino acid residues (predicted Mr = 41,868), a 5' untranslated sequence of 164 bp, a 3' untranslated sequence of 264 bp and a poly(A) tract. The amino acid sequence of the mitochondrial thiolase is 37% identical with that of the mature portion of rat peroxisomal 3-oxoacyl-CoA thiolase precursor. These results suggest that the two thiolases have a common origin and obtained information for targeting to respective organelles during evolution. Two portions in the mitochondrial thiolase that may serve as a mitochondrial targeting signal are presented.     

Regulation of thiolases from pig heart. Control of fatty acid oxidation in heart

The effects of various mitochondrial coenzymes and metabolities on the activities of 3-oxoacyl-CoA thiolase (EC 2.3.1.16) and acetoacetyl-CoA thiolase (EC 2.3.1.9) from pig heart were investigated with the aim of elucidating the possible regulation of these two enzymes. Of the compounds tested, acetyl-CoA was the most effective inhibitor of both thiolases. However, 3-oxoacyl-CoA thiolase was more severly inhibited by acetyl-CoA than was acetoacetyl-CoA thiolase. 3-Oxoacyl-CoA thiolase was also significantly inhibited by decanoyl-CoA while acetoacetyl-CoA thiolase was inhibited by 3-hydroxybutyryl-CoA as strongly as it was by acetyl-CoA. All other compounds either did not affect the thiolase activities or only at unphysiologically high concentrations. The inhibition of acetoacetyl-CoA thiolase by acetyl-CoA was linear and apparently noncompetitive with respect to CoASH (Ki = 125 microM) whereas that of 3-oxoacyl-CoA thiolase was nonlinear. However at low concentrations of acetyl-CoA the inhibition of 3-oxoacyl-CoA thiolase was linear competitive with respect to CoASH (Ki = 3.9 microM). It is concluded that 3-oxoacyl-CoA thiolase, but not acetoacetyl-CoA thiolase, will be completely inhibited by acetyl-CoA at concentrations of CoASH and acetyl-CoA which are assumed to exist intramitochondrially at state-4 respiration. It is suggested that fatty acid oxidation in heart muscle at sufficiently high concentrations of plasma free fatty acids is controlled via the regulation of 3-oxoacyl-CoA thiolase by the acetyl-CoA/CoASH ratio which is determined by the rate of the citric acid cycle and consequently by the energy demand of the tissue.     

The NH2-terminal 14-16 amino acids of mitochondrial and bacterial thiolases can direct mature ornithine carbamoyltransferase into mitochondria

Unlike most mitochondrial matrix proteins, the mitochondrial 3-oxoacyl-CoA thiolase [EC 2.3.1.16] is synthesized with no cleavable presequence and possesses information for mitochondrial targeting and import in the mature protein. This mitochondrial thiolase is homologous with the mature portion of peroxisomal 3-oxoacyl-CoA thiolase and acetoacetyl-CoA thiolase [EC 2.3.1.9] of Zoogloea ramigera along the entire sequence. A hybrid gene encoding the NH2-terminal 16 residues (MALLRGVFIVAAKRTP) of the mitochondrial thiolase fused to the mature portion of rat ornithine carbamoyltransferase [EC 2.1.3.3] (lacking its own presequence) was transfected into COS cells, and subcellular localization of the fusion protein was analyzed. Cell fractionation and immunocytochemical analyses showed that the fusion protein was localized in the mitochondria. These results indicate that the NH2-terminal 16 residues of the mitochondrial thiolase function as a noncleavable signal for mitochondrial targeting and import of this enzyme protein. The fusion protein containing the NH2-terminal 14 residues (MSTPSIVIASARTA) of the bacterial thiolase was also localized in the mitochondria. On the other hand, the fusion protein containing the corresponding portion (MQASASDVVVVHGQRTP) of the peroxisomal thiolase appeared not to be localized to the mitochondria. These results show that the import signal of mitochondrial 3-oxoacyl-CoA thiolase originated from the NH2-terminal portion of the ancestral thiolase. The ancestral enzyme might have already possessed a mitochondrial import activity when mitochondria appeared first, or that it might have acquired the import activity during evolution by accumulation of point mutations in the NH2-terminal portion of the enzyme.     

A noncleavable signal for mitochondrial import of 3-oxoacyl-CoA thiolase

Rat 3-oxoacyl-CoA thiolase, an enzyme of the fatty acid beta-oxidation cycle, is located in the mitochondrial matrix. Unlike most mitochondrial matrix proteins, the thiolase is synthesized with no transient presequence and possesses information for mitochondrial targeting and import in the mature protein of 397 amino acid residues. cDNA sequences encoding various portions of the thiolase were fused in frame to the cDNA encoding the mature portion of rat ornithine transcarbamylase (lacking its own presequence). The fusion genes were transfected into COS cells, and subcellular localization of the fusion proteins was analyzed by cell fractionation with digitonin. When the mature portion of ornithine transcarbamylase was expressed, it was recovered in the soluble fraction. On the other hand, the fusion proteins containing the NH2-terminal 392, 161, or 61 amino acid residues of the thiolase were recovered in the particulate fraction, whereas the fusion protein containing the COOH-terminal 331 residues (residues 62-392) was recovered in the soluble fraction. Enzyme immunocytochemical and immunoelectron microscopic analyses using an anti-ornithine transcarbamylase antibody showed mitochondrial localization of the fusion proteins containing the NH2-terminal portions of the thiolase. These results indicate that the NH2-terminal 61 amino acids of rat 3-oxoacyl-CoA thiolase function as a noncleavable signal for mitochondrial targeting and import of this enzyme protein. Pulse-chase experiments showed that the ornithine transcarbamylase precursor and the thiolase traveled from the cytosol to the mitochondria with half-lives of less than 5 min, whereas the three fusion proteins traveled with half-lives of 10-15 min. Interestingly, in the cells expressing the fusion proteins, the mitochondria showed abnormal shapes and were filled with immunogold-positive crystalloid structures.     

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.     

Evidence for an in vivo modification of mitochondrial proteins by coenzyme A.

Following denaturation of mitochondrial proteins by sodium dodecyl sulfate, a [1-14C]pantothenic acid-derived radioactivity proved to be acid precipitable in the outer membrane, the intermembrane space, the inner membrane and in the matrix of rat liver mitochondria, where it had the highest specific radioactivity of 541 +/- 29 cpm/100 micrograms protein. This tightly and/or covalently bound protein radioactivity could be released by incubation in the presence of dithioerythreitol; it was identified as [14C]coenzyme A by its HPLC retention time, its absorption spectrum and its radioactivity. This acid-stable and thiol-labile coenzyme A-binding apparently refers to specific protein binding sites. With the purified, homogeneous mitochondrial matrix enzymes acetyl-CoA acetyltransferase (acetoacetyl-CoA thiolase) (EC 2.3.1.9, acetyl-CoA:acetyl-CoA C-acetyltransferase) and 3-oxoacyl-CoA thiolase (EC 2.3.1.16) coenzyme A was found exclusively, e.g., in the modified, partially-active forms A1 und A2 of acetyl-CoA acetyltransferase and not in the unmodified fully-active enzyme. Thus it is evident that this coenzyme A modification is transient. We suggest that coenzyme A-modification is a signal involved in the assembly or the degradation process of distinct mitochondrial matrix proteins.     

Identification of [1-14C]pantothenic-acid-mediated modified mitochondrial proteins

The in vivo administration of [1-14C]pantothenic acid, which is the precursor of coenzyme A, resulted in the radioactive labelling of several mitochondrial proteins in rat liver. The incorporated radioactivity could be released by glutathione or 2-mercaptoethanol. Two mitochondrial matrix proteins acetyl-CoA acetyltransferase (liver and heart), an enzyme involved in the biosynthesis or degradation of ketone bodies, and 3-oxoacyl-CoA thiolase (liver), a protein participating in fatty acid oxidation were identified as modified proteins. The radioactivity was localized exclusively in forms A1 and A2 indicating that these forms represent the modified states of the acetyl-CoA acetyltransferase protein. Kinetics of incorporation of radioactivity revealed an accumulation of the modified forms. The ratio of specific radioactivities of A2 compared to A1 was 2.41 +/- 0.15 (n = 10). After in vivo labelling with [14C]leucine, the specific radioactivity of acetyl-CoA acetyltransferase depended on the state of the enzyme protein. The unmodified enzyme exhibited a lower specific radioactivity than its modified forms suggesting different turnover rates of these proteins.     

The oxoacyl-coenzyme A thiolases of animal tissues

1. The activities and relative 3-oxoacyl-CoA substrate specificities of oxoacyl-CoA thiolase were determined in a large number of animal tissues. The relative activities with different 3-oxoacyl-CoA substrates varied widely in different tissues and, in addition, the activity as measured with acetoacetyl-CoA (but not with other longer-carbon-chain acyl-CoA substrates) was activated by K⁺. 2. These properties were due to the presence, in different proportions in each tissue, of three classes of thiolase, all of which use acetoacetyl-CoA as substrate but which have different intracellular locations and substrate specificities and which differ also in kinetic and chromatographic behaviour. 3. Cytoplasmic thiolase activity was found to be widely distributed among different tissues and was due to an acetoacetyl-CoA-specific thiolase. This cytoplasmic activity was found to account for a significant proportion of the total tissue activity towards acetoacetyl-CoA in several tissues, and especially in the brain of newborn rats. 4. Mitochondrial thiolase activity towards acetoacetyl-CoA was due to two different classes of enzyme whose relative amounts varied with the tissue type. An oxoacyl-CoA thiolase of general specificity for the acyl-CoA substrate constituted one class, the other being a specific acetoacetyl-CoA thiolase that differed from its cytoplasmic counterpart in being greatly stimulated by K⁺. 5. This activation by K⁺ made it possible to calculate the tissue contents of mitochondrial acetoacetyl-CoA thiolase and mitochondrial oxoacyl-CoA thiolase from measurements of activity with acetoacetyl-CoA in tissue extracts under defined conditions. 6. The properties and the different thiolases and their tissue distribution is discussed with respect to their possible roles in metabolism.     

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.     

Cloning, expression, and purification of glyoxysomal 3-oxoacyl-CoA thiolase from sunflower cotyledons

The glyoxysomal beta-oxidation system in sunflower (Helianthus annuus L.) cotyledons is distinguished by the coexistence of two different thiolase isoforms, thiolase I and II. So far, this phenomenon has only been described for glyoxysomes from sunflower cotyledons. Thiolase I (acetoacetyl-CoA thiolase, EC 2.3.1.9) recognizes acetoacetyl-CoA only, while thiolase II (3-oxoacyl-CoA thiolase, EC 2.3.1.16) exhibits a more broad substrate specificity towards 3-oxoacyl-CoA esters of different chain length. Here, we report on the cloning of thiolase II from sunflower cotyledons. The known DNA sequence of Cucumis sativus 3-oxoacyl-CoA thiolase was used to generate primers for cloning the corresponding thiolase from sunflower cotyledons. RT-PCR was then used to generate an internal fragment of the sunflower thiolase gene and the termini were isolated using 5'- and 3'-RACE. Full-length cDNA was generated using RT-PCR with sunflower thiolase-specific primers flanking the coding region. The resultant gene encodes a thiolase sharing at least 80% identity with other plant thiolases at the amino acid level. The recombinant sunflower thiolase II was expressed in a bacterial system in an active form and purified to apparent homogeneity in a single step using Ni-NTA agarose chromatography. The enzyme was purified 53.4-fold and had a specific activity of 235 nkat/mg protein. Pooled fractions from the Ni-NTA column resulted in an 83% yield of active enzyme to be used for further characterization.     

Genetic and biochemical heterogeneity in patients with the rhizomelic form of chondrodysplasia punctata — a complementation study

Summary The genetic relationship between 10 patients with clinical manifestations of rhizomelic chondrodysplasia punctata (RCDP) was studied by complementation analysis after somatic cell fusion. Biochemically, 9 out of the 10 patients were characterized by a partial deficiency of acyl-CoA: dihydroxyacetone phosphate acyltransferase (DHAP-AT) and an impairment of plasmalogen biosynthesis, phytanate catabolism and the maturation of peroxisomal 3-oxoacyl-CoA thiolase; 3-oxoacyl-CoA thiolase was strongly reduced in the peroxisomes of these patients. Fusion of fibroblasts from these 9 patients with Zellweger fibroblasts resulted in complementation as indicated by the restoration of DHAP-AT activity, plasmalogen biosynthesis, and punctate fluorescence after staining with a monoclonal antibody to peroxisomal thiolase. No complementation was observed after fusion of different combinations of the 9 RCDP cell lines, suggesting that they belong to a single complementation group. The tenth patient was characterized biochemically by a deficiency of DHAP-AT and an impairment of plasmalogen biosynthesis. However, maturation and localization of peroxisomal thiolase were normal. Fusion of fibroblasts from this patient with fibroblasts from the other 9 patients resulted in complementation as indicated by the restoration of plasmalogen biosynthesis. We conclude that mutations in at least two different genes can lead to the clinical phenotype of RCDP.     

Isolation and subunit composition of native sterol carrier protein 2/3-oxoacyl-coenzyme A thiolase from normal rat liver peroxisomes

In the present report we describe a method for the complete purification of native sterol carrier protein 2/3-oxoacyl-CoA thiolase (SCP-2/thiolase) from normal rat liver peroxisomes. The isolation procedure is based on the alteration in chromatographic properties of the enzyme in the presence of low concentrations of CoA. The purified preparation of SCP-2/thiolase consisted of 58- and 46-kDa polypeptides. Peroxisomes prepared freshly from normal rat liver contained three SCP-2/thiolase isoforms, separable by conventional chromatography. Immunochemical, molecular sieving, and chemical cross-linking experiments indicated that these isoforms represent thiolytically active homo- and heterodimeric combinations of the 46- and 58-kDa subunits (2 x 58, 58-46, and 2 x 46-kDa proteins).     

Type-II 3-oxoacyl-CoA thiolase of the nematode Caenorhabditis elegans is located in peroxisomes, highly expressed during larval stages and induced by clofibrate.

We examined the expression and localization of type-II 3-oxoacyl-CoA thiolase in the nematode Caenorhabditis elegans. Type-II thiolase acts on 3-oxoacyl-CoA esters with a methyl group at the alpha carbon, whereas conventional thiolases do not. Mammalian type-II thiolase, which is also termed sterol carrier protein x (SCPx) or SCP2/3-oxoacyl-CoA thiolase, is located in the peroxisomes and involved in phytanic acid degradation and most probably in bile acid synthesis. The nematode enzyme lacks the SCP2 domain, which carries the peroxisomal-targeting signal, but produces bile acids in a cell-free system. Northern and Western blot analyses demonstrated that C. elegans expressed type-II thiolase throughout its life cycle, especially during the larval stages, and that the expression was significantly enhanced by the addition of clofibrate at 5 mM or more to the culture medium. Whole-mount in situ hybridization and immunostaining of L4 larvae revealed that the enzyme was mainly expressed in intestinal cells, which are multifunctional like many of the cell types in C. elegans. Subcellular fractionation and indirect immunoelectron microscopy of the nematode detected the enzyme in the matrix of peroxisomes. These results indicate the fundamental homology between mammalian SCPx and the nematode enzyme regardless of whether the SCP2 part is fused, suggesting their common physiological roles.     

Peroxisomes of normal morphology but deficient in 3-oxoacyl-CoA thiolase in rhizomelic chondrodysplasia punctata fibroblasts.

Rhizomelic Chondrodysplasia Punctata (RCDP) is an autosomal recessive disorder in which plasmalogen biosynthesis and phytanate catabolism are impaired. Peroxisomal structure and the intracellular localization of catalase, the 69 kDa peroxisomal integral membrane protein (PMP), and 3-oxoacyl-CoA thiolase were studied in cultured skin fibroblasts from control subjects and patients with RCDP. A punctate fluorescence pattern characteristic for peroxisomes was seen in control cells incubated with either anti-(catalase), anti-(69 kDa PMP) or anti-(3-oxoacyl-CoA thiolase). Incubation of mutant cells with anti-(catalase) or anti-(69 kDa PMP) resulted in the same pattern. However, when RCDP fibroblasts were incubated with a monoclonal anti-(3-oxoacyl-CoA thiolase) antibody no punctate fluorescence could be observed. Cryosections from control and RCDP cells were examined by electron microscopy using double immunogold labelling. RCDP fibroblasts contained structures indistinguishable from control peroxisomes, the membranes reacting with anti-(69 kDa PMP) and the matrix with anti-(catalase). However, the matrix of RCDP peroxisomes, unlike control peroxisomes, did not react with anti-(3-oxoacyl-CoA thiolase). We conclude that RCDP fibroblasts contain regularly shaped peroxisomes, comparable to control peroxisomes in number as well as in content of catalase and 69 kDa PMP. However, in RCDP peroxisomes the amount of 3-oxoacyl-CoA thiolase protein proved to be below the limit of detection.     

Human trifunctional protein deficiency: a new disorder of mitochondrial fatty acid beta-oxidation.

In this paper we report the identification of a new disorder of mitochondrial fatty acid beta-oxidation in a patient which presented with clear manifestations of a mitochondrial beta-oxidation disorder. Subsequent studies in fibroblasts revealed an impairment in palmitate beta-oxidation and in addition, a combined deficiency of long-chain enoyl-CoA hydratase, long-chain 3-hydroxyacyl-CoA-dehydrogenase and long-chain 3-oxoacyl-CoA thiolase. The recent identification of a multifunctional, membrane-bound beta-oxidation enzyme protein catalyzing all these three enzyme activities (Carpenter et al. (1992) Biochem. Biophys. Res. Commun. 183, 443-448; Uchida et al. (1992) J. Biol. Chem. 267, 1034-1041) suggested an underlying basis for this peculiar combination of three enzyme deficiencies. We show by means of size-exclusion chromatography that there is, indeed, a deficiency of the multifunctional beta-oxidation enzyme protein in this patient.     

Peroxisomes of normal morphology but deficient in 3-oxoacyl-CoA thiolase in Rhizomelic Chondrodysplasia Punctata fibroblasts

Rhizomelic Chondrodysplasia Punctata (RCDP) is an autosomal recessive disorder in which plasmalogen biosynthesis and phytanate catabolism are impaired. Peroxisomal structure and the intracellular localization of catalase, the 69 kDa peroxisomal integral membrane protein (PMP), and 3-oxoacyl-CoA thiolase were studied in cultured skin fibroblasts from control subjects and patients with RCDP. A punctate fluorescence pattern characteristic for peroxisomes was seen in control cells incubated with either anti-(catalase), anti-(69 kDa PMP) or anti-(3-oxoacyl- CoA thiolase). Incubation of mutant cells with anti-(catalase) or anti-(69 kDa PMP) resulted in the same pattern. However, when RCDP fibroblasts were incubated with a monoclonal anti-(3-oxoacyl-CoA thiolase) antibody no punctate fluorescence could be observed. Cryosections from control and RCDP cells were examined by electron microscopy using double immunogold labelling. RCDP fibroblasts contained structures indistinguishable from control peroxisomes, the membranes reacting with anti-(69 kDa PMP) and the matrix with anti-(catalase). However, the matrix of RCDP peroxisomes, unlike control peroxisomes, did not react with anti-(3-oxoacyl-CoA thiolase). We conclude that RCDP fibroblasts contain regularly shaped peroxisomes, comparable to control peroxisomes in number as well as in content of catalase and 69 kDa PMP. However, in RCDP peroxisomes the amoung of 3-oxoacyl-CoA thiolase protein proved to be below the limit of detection.     

Biosynthesis of enzymes of rat-liver mitochondrial beta-oxidation

The biogenesis of seven enzymes involved in the mitochondrial fatty acid beta-oxidation of rat liver was studied. Hepatic RNA was translated in vitro in a rabbit reticulocyte lysate cell-free system and the translation products were immunoprecipitated, subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis and visualized by fluorography. The translation products obtained in vitro of medium-chain and/or long-chain acyl-CoA dehydrogenase (these enzymes were immunochemically cross-reactive), enoyl-CoA hydratase, 3-hydroxyacyl-CoA dehydrogenase, and acetoacetyl-CoA thiolase and probably also short-chain acyl-CoA dehydrogenase were larger than the subunits of the corresponding mature enzymes by 2-4.5 kDa, whereas the 3-oxoacyl-CoA thiolase obtained in vitro was approximately the same size as the mature subunit. The free polysome fraction of rat liver was 4.3-9.0-times more active than the membrane-bound polysome fraction in the synthesis of these seven enzymes. The enzyme activities were increased after administration of di(2-ethylhexyl)phthalate; the extent of the increase varied from one enzyme to another. The increase in the cell-free translation activity of total hepatic RNA for these enzymes after administration of the chemical was markedly different among individual enzymes and higher than that in the rates of synthesis of the corresponding enzymes which were determined by the experiment in vivo.     

4-Bromo-2-octenoic acid specifically inactivates 3-ketoacyl-CoA thiolase and thereby fatty acid oxidation in rat liver mitochondria

In an attempt to develop a compound which would specifically inhibit 3-ketoacyl-CoA thiolase (EC 2.3.1.16) in whole mitochondria, 4-bromo-2-octenoic acid was synthesized and studied. After rat liver mitochondria were preincubated with 4-bromo-2-octenoic acid for 3 min, respiration supported by either palmitoylcarnitine or pyruvate was completely abolished, whereas no inhibition was observed with rat heart mitochondria. Addition of carnitine stimulated respiration supported by pyruvate without relieving inhibition of palmitoylcarnitine-dependent respiration. Hence, this compound seems to be a specific inhibitor of beta-oxidation. When the enzymes of beta-oxidation were assayed in a soluble extract prepared from mitochondria preincubated with 4-bromo-2-octenoic acid, only 3-ketoacyl-CoA thiolase was found to be inactivated. 4-Bromo-2-octenoic acid is metabolized by mitochondrial beta-oxidation enzymes to 3-keto-4-bromooctanoyl-CoA which effectively and irreversibly inhibits 3-ketoacyl-CoA thiolase but not acetoacetyl-CoA thiolase (EC 2.3.1.9). Even though 3-keto-4-bromooctanoyl-CoA inhibits the latter enzyme reversibly, 4-bromo-2-octenoic acid does not inhibit ketogenesis in rat liver mitochondria with acetylcarnitine as a substrate. It is concluded that 4-bromo-2-octenoic acid specifically inhibits mitochondrial fatty acid oxidation by inactivating 3-ketoacyl-CoA thiolase in rat liver mitochondria.