A novel type of short- and medium-chain acyl-CoA hydrolases in brown adipose tissue mitochondria

Acyl-CoA hydrolase activities were studied in brown adipose tissue from hamsters. A latent activity was observed in isolated mitochondria. Two peaks of activity were clearly visible in mitochondria, one with an optimum at propionyl-CoA ("short-chain hydrolase") and one with an optimum at nonanoyl-CoA ("medium-chain hydrolase"); there was only low activity toward palmitoyl-CoA and longer-chain acyl-CoAs. In subcellular fractionation experiments, the activity of the short-chain and the medium-chain hydrolase fully followed that of the mitochondrial matrix marker enzyme 2-oxoglutarate dehydrogenase. The specific activity of the hydrolases in the mitochondrial fraction was doubled after cold acclimation. beta-NADH inhibited the short- and medium-chain hydrolases; alpha-NADH, NADPH, and NAD+ were without effect. ADP stimulated the short- and medium-chain hydrolases; ATP and AMP were practically without effect. Evidence is presented to indicate that NADH and ADP interact on the enzyme at the same site and that ADP is essential for the maintenance of the short- and medium-chain enzyme activities. A positive effect of KCl was found on the short- and medium-chain hydrolase activities. Also, the divalent ions Ca2+ and Mg2+ were stimulatory, but only Ca2+ was able to overcome NADH inhibition, possibly due to interaction directly with NADH. It is concluded that brown adipose tissue mitochondria, besides a conventional type of acyl-CoA hydrolase, contain two species of a novel type of acyl-CoA hydrolases which are characterized by being regulated by ADP and NADH (interacting at a common site) and by having an obligatory requirement for ADP.     

The presence of acyl-CoA hydrolase in rat brown-adipose-tissue peroxisomes.

The subcellular distribution of acyl-CoA hydrolase was studied in rat brown adipose tissue, with special emphasis on possible peroxisomal localization. Subcellular fractionation by sucrose-density-gradient centrifugation, followed by measurement of short-chain (propionyl-CoA) acyl-CoA hydrolase in the presence of NADH, resulted in two peaks of activity in the gradient: one peak corresponded to the distribution of cytochrome oxidase (mitochondrial marker enzyme), and another peak of activity coincided with the peroxisomal marker enzyme catalase. The distribution of the NADH-inhibited short-chain hydrolase activity fully resembled that of cytochrome oxidase. The substrate-specificity curve of the peroxisomal acyl-CoA hydrolase activity indicated the presence of a single enzyme exhibiting a broad substrate specificity, with maximal activity towards fatty acids with chain lengths of 3-12 carbon atoms. The mitochondrial acyl-CoA hydrolase substrate specificity, in contrast, indicated the presence of at least two acyl-CoA hydrolases (of short- and medium-chain-length specificity). The peroxisomal acyl-CoA hydrolase activity was inhibited by CoA at low (microM) concentrations and by ATP at high concentrations (greater than 0.8 mM). In contrast with the mitochondrial short-chain hydrolase, the peroxisomal acyl-CoA hydrolase activity was not inhibited by NADH.     

Effects of clofibric acid and tiadenol on cytosolic long-chain acyl-CoA hydrolase and peroxisomal beta-oxidation in liver and extrahepatic tissues of rats

The effects of two peroxisome proliferators, p-chlorophenoxyisobutyric acid (clofibric acid) and 2,2'-(decamethylenedithio)diethanol (tiadenol), on cytosolic long-chain acyl-CoA hydrolase and peroxisomal beta-oxidation were studied in several organs of rat. Among organs of control rats, the brain had the highest activity of long-chain acyl-CoA hydrolase, followed by testis, and a low activity was found in other tissues. Administration of the peroxisome proliferators caused a marked increase in activity of long-chain acyl-CoA hydrolase in both liver and intestinal mucosa and a slight increase in the activity in kidney, but little affected acyl-CoA hydrolase activity in either brain, testis, heart, spleen and skeletal muscle. In accordance with the change in the activity of acyl-CoA hydrolase, the activity of peroxisomal beta-oxidation was markedly increased in liver, intestinal mucosa and kidney, and a slight increase was found in brain and testis, whereas peroxisome proliferators little affected the activity in other organs tested. Gel filtration of cytosol from intestinal mucosa showed that clofibric acid caused an appearance of a new peak in intestinal mucosa. Although cytosol of liver, intestinal mucosa, brain and testis contained two 4-nitrophenyl acetate esterases with different molecular weights (about 105,000 and about 55,000), these esterases are different from cytosolic long-chain acyl-CoA hydrolases of these four organs in respect of molecular weight. The administration of clofibric acid little affected cytosolic 4-nitrophenyl acetate esterases. Comparative studies on cytosolic long-chain acyl-CoA hydrolases from these four organs showed that liver hydrolase I (molecular weight of about 80,000) had properties similar to those of brain and testis enzymes. On the other hand, intestinal mucosa enzyme was different from either hepatic hydrolase I or II (molecular weight of about 40,000). The results from the present study suggest that inductions of peroxisomal beta-oxidation and cytosolic long-chain acyl-CoA hydrolases are essential responses of rats to peroxisome proliferators not only in liver but also in intestinal mucosa and that induced hydrolases are not attributable to non-specific esterases.     

Long-chain acyl-CoA hydrolase in the brain

Summary. Long-chain acyl-CoA hydrolases are a group of enzymes that cleave acyl-CoAs into fatty acids and coenzyme A (CoA-SH). Because acyl-CoAs participate in numerous reactions encompassing lipid synthesis, energy metabolism and regulation, modulating intracellular levels of acyl-CoAs would affect cellular functions. Therefore, acyl-CoA synthetases have been intensively studied. In contrast, acyl-CoA hydrolases have been less investigated, especially in the brain despite the fact that its long-chain acyl-CoA hydrolyzing activity is much higher than that in any other organ in the body. However, recent studies have dissected the multiplicity of this class of enzymes on a genomic basis, and have allowed us to discuss their function. Here, we describe a cytosolic long-chain acyl-CoA hydrolase (referred to as BACH) that is constitutively expressed in the brain, comparing it with other acyl-CoA hydrolases found in peripheral organs that have a role in fatty acid oxidation.     

Acylation of 2-acyl-glycerophosphocholine in guinea-pig heart microsomal fractions.

Acyl-CoA:2-acyl-sn-glycero-3-phosphocholine (GPC) acyltransferase is required for the maintenance of the asymmetric distribution of saturated fatty acids at the C-1 position of phosphatidylcholine; however, this activity has been reported to be absent in cardiac tissue. In the present study a very active acyl-CoA:2-acyl-GPC activity was detected and characterized in guinea-pig heart microsomes (microsomal fractions); the mitochondria did not appear to possess this activity. The acyl-CoA specificity of the microsomal acyl-CoA:2-acyl-GPC acyltransferase was distinct from the corresponding acyl-CoA:1-acyl-GPC acyltransferase. These differences were due to the position of the fatty acid on the lysophospholipid rather than the composition of the fatty acids. The enzyme did not exhibit a distinct preference for saturated fatty acids, as might be expected. Our results suggest that, in the heart, control of the intracellular composition and concentration of acyl-CoAs by acyl-CoA hydrolase and acyl-CoA synthetase may play an important role in maintaining the asymmetric distribution of fatty acids in phosphatidylcholine.     

Sex-related difference in the effect of clofibric acid on induction of two novel long-chain acyl-CoA hydrolases in rat liver

The sex differences in the induction of two novel long-chain acyl-CoA hydrolases in hepatic cytosol of rats by clofibric acid (p-chlorophenoxyisobutyric acid)-feeding and the properties of the induced acyl-CoA hydrolases were investigated. Marked sex-related difference was observed in the induction of acyl-Coa hydrolase activity. The sex difference was mainly due to the difference in the induction of acyl-CoA hydrolase with higher molecular weight (hydrolase I), but not to the difference in the induction of acyl-CoA hydrolase with lower molecular weight (hydrolase II). The extent of the induction of the hydrolase I in hepatic cytosol of male rats was 3.5 times over that of female rats. Castration of male rats resulted in the marked depression of the ability to induce hydrolase I. The administration of testosterone to the castrated male rats recovered completely the ability to induce hydrolase I. Unlike hydrolase I, the ability to induce hydrolase II did not respond to the changes in state of androgen. The administration of di-(2-ethylhexyl)phthalate also induced both hydrolase I and II, although the extent of the induction of hydrolase I was less compared to that by clofibric acid treatment. Likewise, marked sex difference was observed in the induction of the hydrolase I on di-(2-ethylhexyl)phthalate administration. These two hydrolases showed different kinetic properties and different substrate specificities to each other. Hydrolase I was inhibited by bovine serum albumin in vitro, but was not affected by Mg2+. Hydrolase II was activated slightly in the presence of lower concentrations of bovine serum albumin, Mg2+ or Ca2+.     

Intracellular localization of long-chain acyl-coenzyme A hydrolase and acyl-L-carnitine hydrolase in brown adipose tissue from guinea pigs.

The activities of long-chain acyl-CoA hydrolase (palmitoyl-CoA hydrolase, EC 3.1.2.2) and long-chain acyl-L-carnitine hydrolase, EC 3.1.1.28) in brown adipose tissue from cold-exposed and control guinea pigs were studied. Mitochondria from cold-exposed animals hydrolysed 21 nmol of palmitoyl-CoA/min per mg of protein and 1.3 nmol of palmitoyl-L-carnitine/min per mg of protein, and the specific activities were respectively 2 and 5 times as high in cold-exposed as in control animals. The subcellular-localization studies showed that both the long-chain acyl-CoA hydrolase and long-chain acyl-L-carnitine hydrolase were localized in the mitochondria. A location also in the soluble fraction cannot be excluded. The long-chain acyl-CoA hydrolase activity was doubled when the mitochondria were disrupted; this indicates that the enzyme is localized in the matrix compartment.     

Quantitation of the efflux of acylcarnitines from rat heart, brain, and liver mitochondria

The efflux of individual short-chain and medium-chain acylcarnitines from rat liver, heart, and brain mitochondria metabolizing several substrates has been measured. The acylcarnitine efflux profiles depend on the substrate, the source of mitochondria, and the incubation conditions. The largest amount of any acylcarnitine effluxing per mg of protein was acetylcarnitine produced by heart mitochondria from pyruvate. This efflux of acetylcarnitine from heart mitochondria is almost 5 times greater with 1 mM than 0.2 mM carnitine. Apparently the acetyl-CoA generated from pyruvate by pyruvate dehydrogenase is very accessible to carnitine acetyltransferase. Very little acetylcarnitine effluxes from heart mitochondria when octanoate is the substrate except in the presence of malonate. Acetylcarnitine production from some substrates peaks and then declines, indicating uptake and utilization. The unequivocal demonstration that considerable amounts of propionylcarnitine or isobutyrylcarnitine efflux from heart mitochondria metabolizing alpha-ketoisovalerate and alpha-keto-beta-methylvalerate provides evidence for a role (via removal of non-metabolizable propionyl-CoA or slowly metabolizable acyl-CoAs) for carnitine in tissues which have limited capacity to metabolize propionyl-CoA. These results also show propionyl-CoA must be formed during the metabolism of alpha-ketoisovalerate and that extra-mitochondrial free carnitine rapidly interacts with matrix short-chain aliphatic acyl-CoA generated from alpha-keto acids of branched-chain amino acids and pyruvate in the presence and absence of malate.     

Hepatic enzymatic synthesis and hydrolysis of CoA esters of solvent-derived oxa acids

Many ethylene glycol-derived solvents are oxidized to xenobiotic alkoxyacetic acids (3-oxa acids) by hepatic enzymes. The toxicity of these ubiquitous solvents has been associated with their oxa acid metabolites. For many xenobiotic carboxylic acids, the toxicity is associated with the CoA ester of the acid. In this study, related alkoxyacetic acids were evaluated as potential substrates for acyl-CoA synthetases found in mitochondrial, peroxisomal, and microsomal fractions isolated from rat liver. Likewise, chemically synthesized oxa acyl-CoAs were used as substrates for acyl-CoA hydrolases associated with the same rat liver fractions. Activities of the xenobiotic oxygen-substituted substrates were compared with analogous physiologic aliphatic substrates by UV-vis spectrophotometric methods. All of the solvent-derived oxa acids were reasonable substrates for the acyl-CoA synthetases, although their activity was usually less than the corresponding physiologic acid. Acyl-CoA hydrolase activities were decreased compared with acyl-CoA synthetase activities for all substrates, especially for the oxa acyl-CoAs. These studies suggest that these xenobiotic carboxylic acids may be converted to reactive acyl-CoA moieties which will persist in areas of the cell proximal to lipid synthesis, beta-oxidation, protein acylation, and amino acid conjugation. The interaction of these xenobiotic acyl-CoAs with those processes may be important to their toxicity and/or detoxification.     

Purification and characterization of the 2-methyl branched-chain Acyl-CoA dehydrogenase, an enzyme involved in NADH-dependent enoyl-CoA reduction in anaerobic mitochondria of the nematode, Ascaris suum

The 2-methyl branched-chain acyl-CoA dehydrogenase was purified to homogeneity from mitochondria of the parasitic nematode, Ascaris suum. The native molecular weight of the enzyme was estimated to be 170,000 by gel filtration. The enzyme migrated as a single protein band with Mr = 42,500 during sodium dodecyl sulfate-polyacrylamide gel electrophoresis suggesting that the enzyme is a tetramer composed of identical subunits. The enzyme exhibited absorbance maxima at 272, 375, and 452 with a ratio 7.9:0.8:1.0, respectively. FAD content was estimated to be 0.9 mol/mol of subunit and the absorption coefficient of FAD at 452 nm was 14.1 mM-1 cm-1. The purified enzyme dehydrogenated both 2-methylbutyryl-CoA and 2-methylvaleryl-CoA with apparent Km and Vmax values of 18 microM and 1.62 mumol/min/mg and 21 microM and 1.58 mumol/min/mg, respectively. This enzyme also appeared to dehydrogenate butyryl-CoA, valeryl-CoA, and octanoyl-CoA but at a much lower rate. The enzyme did not dehydrogenate propionyl-CoA, isobutyryl-CoA, isovaleryl-CoA, and palmitoyl-CoA. Tiglyl-CoA and 2-methyl-2-pentenoyl-CoA were identified as reaction products from 2-methylbutyryl- and 2-methylvaleryl-CoA, respectively. Dehydrogenating activity with both substrates was inhibited by tiglyl-CoA, acetoacetyl-CoA, and straight chain acyl CoAs of increasing chain length. N-Ethylmaleimide and p-hydroxymercuribenzoate had little effect on dehydrogenating activity but the heavy metals Hg2+ and Ag2+ were potent inhibitors. Physiologically, the dehydrogenase functions as a branched-chain enoyl-CoA reductase. Incubations of A. suum submitochondrial particles, NADH, tiglyl-CoA, purified A. suum electron-transfer flavoprotein, and the 2-methyl branched-chain acyl-CoA dehydrogenase resulted in the rotenone-sensitive, dehydrogenase-dependent formation of 2-methylbutyryl-CoA.     

Purification and characterization of 2-methyl-branched chain acyl coenzyme A dehydrogenase, an enzyme involved in the isoleucine and valine metabolism, from rat liver mitochondria

2-Methyl-branched chain acyl-CoA dehydrogenase was purified to homogeneity from rat liver mitochondria. The native molecular weight of the enzyme was estimated to be 170,000 by gel filtration. On sodium dodecyl sulfate-polyacrylamide gel electrophoresis both with and without 2-mercaptoethanol, the enzyme showed a single protein band with Mr = 41,500, suggesting that this enzyme is composed of four subunits of equal size. Its isoelectric point was 5.50 +/- 0.2, and A1%280 nm was 12.5. This enzyme contained protein-bound FAD. The purified enzyme dehydrogenated S-2-methylbutyryl-CoA and isobutyryl-CoA with equal activity. The activities with each of these compounds were co-purified throughout the entire purification procedure. This enzyme also dehydrogenated R-2-methylbutyryl-CoA, but the specific activity was considerably lower (22%) than that for the S-enantiomer. The enzyme did not dehydrogenate other acyl-CoAs, including isovaleryl-CoA, propionyl-CoA, butyryl-CoA, octanoyl-CoA, and palmitoyl-CoA, at any significant rate. Apparent Km and Vmax values for S-2-methylbutyryl-CoA were 20 microM and 2.2 mumol min-1 mg-1, respectively, while those for isobutyryl-CoA were 89 microM and 2.0 mumol min-1 mg-1 using phenazine methosulfate as an artificial electron acceptor. The enzyme was also active with electron transfer flavoprotein. Tiglyl-CoA and methacrylyl-CoA were identified as the reaction products from S-2-methylbutyryl-CoA and isobutyryl-CoA, respectively. 2-Ethylacrylyl-CoA was produced from R-2-methylbutyryl-CoA. Tiglyl-CoA competitively inhibited the activity with both S-2-methylbutyryl-CoA and isobutyryl-CoA with a similar Ki. The enzyme activity was also severely inhibited by several organic sulfhydryl reagents such as N-ethylmaleimide, p-hydroxymercuribenzoate, and methyl mercury iodide. The pattern and degree of inhibition were essentially identical for both substrates. The purified 2-methyl-branched chain acyl-CoA dehydrogenase was immunologically distinct from isovaleryl-CoA-, short chain acyl-CoA-, medium chain acyl-CoA-, or long chain acyl-CoA dehydrogenase.     

Microsomal fatty acyl-CoA transacylation and hydrolysis: fatty acyl-CoA species dependent modulation by liver fatty acyl-CoA binding proteins1

Liver and intestinal cytosol contain abundant levels of long chain fatty acyl-CoA binding proteins such as liver fatty acid binding protein (L-FABP) and acyl-CoA binding protein (ACBP). However, the relative function and specificity of these proteins in microsomal utilization of long chain fatty acyl-CoAs (LCFA-CoAs) for sequential transacylation of glycerol-3-phosphate to form phosphatidic acid is not known. The results showed for the first time that L-FABP and ACBP both stimulated microsomal incorporation of the monounsaturated oleoyl-CoA and polyunsaturated arachidonoyl-CoA 8–10-fold and 2–3-fold, respectively. In contrast, these proteins inhibited microsomal utilization of the saturated palmitoyl-CoA by 69% and 62%, respectively. These similar effects of L-FABP and ACBP on microsomal phosphatidic acid biosynthesis were mediated primarily through the activity of glycerol-3-phosphate acyltransferase (GPAT), the rate limiting step, rather than by protecting the long chain acyl-CoAs from microsomal hydrolase activity. In fact, ACBP but not L-FABP protected long chain fatty acyl-CoAs from microsomal acyl-CoA hydrolase activity in the order: palmitoyl-CoA>oleoyl-CoA>arachidonoyl-CoA. In summary, the data established for the first time a role for both L-FABP and ACBP in microsomal phosphatidic acid biosynthesis. By preferentially stimulating microsomal transacylation of unsaturated long chain fatty acyl-CoAs while concomitantly exerting their differential protection from microsomal acyl-CoA hydrolase, L-FABP and ACBP can uniquely function in modulating the pattern of fatty acids esterified to phosphatidic acid, the de novo precursor of phospholipids and triacylglycerols. This may explain in part the simultaneous presence of these proteins in cell types involved in fatty acid absorption and lipoprotein secretion.     

Purification and characterisation of leukotriene A4 hydrolase from rat neutrophils

Leukotriene A4 hydrolase was rapidly and extensively purified from rat neutrophils using anion exchange and gel filtration high-pressure liquid chromatography. The enzyme which converts the allylic epoxide leukotriene A4 to the 5,12-dihydroxyeicosatetraenoic acid leukotriene B4 was localized in the cytosolic fraction and exhibited an optimum activity at pH 7.8 and an apparent Km for leukotriene A4 between 2 X 10(-5) and 3 X 10(-5) M. The purified leukotriene A4 hydrolase was shown to have a molecular weight of 68 000 on sodium dodecylsulfate polyacrylamide gel electrophoresis and of 50 000 by gel filtration. The molecular weight and monomeric native form of this enzyme are unique characteristics which distinguish leukotriene A4 hydrolase from previously purified epoxide hydrolases.     

Purification, molecular cloning, and genomic organization of human brain long-chain acyl-CoA hydrolase

An acyl-CoA hydrolase, referred to as hBACH, was purified from human brain cytosol. The enzyme had a molecular mass of 100 kDa and 43-kDa subunits, and was highly active with long-chain acyl-CoAs, e.g. a maximal velocity of 295 micromol/min/mg and K(m) of 6.4 microM for palmitoyl-CoA. Acyl-CoAs with carbon chain lengths of C(8-18) were also good substrates. In human brain cytosol, 85% of palmitoyl-CoA hydrolase activity was titrated by an anti-BACH antibody, which accounted for over 75% of the enzyme activity found in the brain tissue. The cDNA isolated for hBACH, when expressed in Escherichia coli, directed the expression of palmitoyl-CoA hydrolase activity and a 44-kDa protein immunoreactive to the anti-BACH antibody, which in turn neutralized the hydrolase activity. The hBACH cDNA encoded a 338-amino acid sequence which was 95% identical to that of a rat homolog. The hBACH gene spanned about 130 kb and comprised 9 exons, and was mapped to 1p36.2 on the cytogenetic ideogram. These findings indicate that the long-chain acyl-CoA hydrolase present in the brain is well conserved between man and the rat, suggesting a conserved role for this enzyme in the mammalian brain, and enabling genetic studies on the functional analysis of acyl-CoA hydrolase.     

Partial purification and properties of acyl-CoA reductase from Clostridium butyricum.

A long chain acyl-CoA reductase of Clostridium butyricum has been partially purified from the 100,000g supernatant fraction of cell extracts. The enzyme reduces acyl-CoA derivatives to aldehydes in the presence of NADH. It is stable in dithiothreitol-containing buffers at 4 °C, heat-labile, and sensitive to sulfhydryl reagents. It is active with palmitoyl-CoA, stearoyl-CoA, oleoyl-CoA, and myristoyl-CoA. Its apparent molecular weight on Sephadex G-100 column chromatography is 50,000. In crude extracts and at low purification, an NADPH-dependent reduction of palmitaldehyde to cetyl alcohol was also observed. An acyl-CoA hydrolase was also observed in crude extracts.     

Acyl-CoA pool and acyl-CoA thioesterase in Streptomyces noursei var. polifungini.

The size of acyl-CoA pool in S. noursei var. polifungini was found to be associated with the antibiotic-synthesizing ability, and was negatively correlated with both the thio-esterase activity (acetyl-CoA hydrolase, EC 3.1.2.1) and its affinity towards acetyl-CoA and propionyl-CoA. The apparent Michaelis constants with acetyl-CoA and propionyl-CoA were 33 times 10(-5) and 6.6 times 10(-5) M in the low producing strain, and 8.5 times 10(-4) and 1.2 times 10(-4) M in the high producing strain, respectively.     

Long-chain acyl-CoA oxidases of Arabidopsis

Full-length cDNAs coding for two distinct acyl-CoA oxidases were isolated by screening an Arabidopsis cDNA library. The genes for the two acyl-CoA oxidases have been termed AtACX1 and AtACX2. AtACX1 encodes a peptide of 664 amino acids possessing a molecular mass of 74.3 kDa. AtACX2 encodes a peptide of 691 amino acids in length with a molecular mass of 77.5 kDa. Peroxisomal targeting signals were identified in the primary sequences. AtACX1 has a putative PTS1, whereas AtACX2 has a characteristic PTS2. Expression of AtACX1 and AtACX2 in Escherichia coli gave active enzymes for enzymatic and biochemical analysis. AtACX1 was active with both medium-and long-chain saturated fatty acyl-CoAs and showed maximal activity with C14-CoA. Activity with mono-unsaturated acyl-CoAs was slightly higher than with the corresponding saturated acyl-CoA. AtACX2 was active with long-chain acyl-CoAs and showed maximal activity with C18-CoA. AtACX2 activity with mono-unsaturated acyl-CoAs was approximately twice as high as with the corresponding saturated acyl-CoA. Both enzymes have an apparent Km of approximately 5 microM with the preferred substrate. Northern analysis was conducted to determine the expression patterns of AtACX1 and AtACX2 during germination and in various tissues of a mature plant. The two genes showed generally similar expression profiles and steady-state mRNA levels in seedlings and mature tissues, but subtle differences were observed. Enzymatic analyses of plant extracts revealed that AtACX1 and AtACX2 are members of a family that includes acyl-CoA oxidases specific for shorter-chain acyl-CoAs. Through expression of antisense constructs of the individual genes, we were able to decrease long-chain oxidase activity only in antisense AtACX1 plants. Seedlings with long-chain oxidase activity reduced down to 30% of wild-type levels germinated and established normally; however, reduced root growth appeared to be a general feature of antisense AtACX1 plants.     

Quantitation of the effect of L-carnitine on the levels of acid-soluble short-chain acyl-CoA and CoASH in rat heart and liver mitochondria

The steady state levels of mitochondrial acyl-CoAs produced during the oxidation of pyruvate, alpha-ketoisovalerate, alpha-ketoisocaproate, and octanoate during state 3 and state 4 respiration by rat heart and liver mitochondria were determined. Addition of carnitine lowered the amounts of individual short-chain acyl-CoAs and increased CoASH in a manner that was both tissue- and substrate-dependent. The largest effects were on acetyl-CoA derived from pyruvate in heart mitochondria using either state 3 or state 4 oxidative conditions. Carnitine greatly reduced the amounts of propionyl-CoA derived from alpha-ketoisovalerate, while smaller effects were obtained on the branched-chain acyl-CoA levels, consistent with the latter acyl moieties being poorer substrates for carnitine acetyltransferase and also poorer substrates for the carnitine/acylcarnitine translocase. The levels of acetyl-CoA in heart and liver mitochondria oxidizing octanoate during state 3 respiration were lower than those obtained with pyruvate. The rate of acetylcarnitine efflux from heart mitochondria during state 3 (with pyruvate or octanoate as substrate, in the presence or absence of malate with 0.2 mM carnitine) shows a linear response to the acetyl-CoA/CoASH ratio generated in the absence of carnitine. This relationship is different for liver mitochondria. These data demonstrate that carnitine can modulate the aliphatic short-chain acyl-CoA/CoA ratio in heart and liver mitochondria and indicate that the degree of modulation varies with the aliphatic acyl moiety.     

Purification and Some Properties of Maleylpyruvate Hydrolase and Fumarylpyruvate Hydrolase from Pseudomonas alcaligenes

Hydrolysis of the gentisate ring-cleavage product, maleylpyruvate (cis-2,4-diketohept-5-enedioic acid), was shown to be catalyzed by an enzyme, maleylpyruvate hydrolase 11, in Pseudomonas alcaligenes (P25X1) after growth with 3-hydroxybenzoate. This activity was separated from fumarylpyruvate hydrolase activity during the course of its purification which accomplished an approximately 50-fold increase in specific activity. An apparent molecular weight of 77,000 was assigned on the basis of Sephadex G-200 chromatography. Despite the presence of up to three similarly migrating bands of protein on polyacrylamide-gel electrophoresis of the purified enzyme, at least two of these bands possessed maleylpyruvate hydrolase activity. Electrophoresis on sodium dodecyl sulfate-polyacrylamide before and after reduction with mercaptoethanol gave a principal band of molecular weight of 33,000 (and a minor band of molecular weight 50,000). A number of substituted maleylpyruvates also served as substrates for maleylpyruvate hydrolase 11, but maleylacetoacetate and fumarylpyruvate were not attacked. Fumarylpyruvate hydrolase was purified approximately 40-fold to give a single band on polyacrylamide gels and with an apparent molecular weight of 73,000 by Sephadex G-200 chromatography. Upon sodium dodecyl sulfate-polyacrylamide gel electrophoresis before or after reduction with mercaptoethanol, a subunit molecular weight of 25,000 was obtained. Neither maleylpyruvate nor fumarylacetoacetate served as substrates for fumarylpyruvate hydrolase. The activities of both maleyl- and fumarylpyruvate hydrolases were stimulated by Mn²⁺ ions. Reasons are discussed for the presence of both enzyme activities, one of which appears to be redundant.     

Properties of purified carnitine acyltransferases of mouse liver peroxisomes

The purpose of this study was to characterize the physical, kinetic, and immunological properties of carnitine acyltransferases purified from mouse liver peroxisomes. Peroxisomal carnitine octanoyltransferase and carnitine acetyltransferase were purified to apparent homogeneity from livers of mice fed a diet containing the hypolipidemic drug Wy-14,643 [( 4-chloro-6-(2,3-xylidino)-2-pyrimidinylthio]-acetic acid). Both enzymes have a molecular weight of 60,000 and a similar pH optimum. Carnitine octanoyltransferase had a maximum activity for C6 moieties while the maximum for carnitine acetyltransferase was with C3 and C4 moieties. The apparent Km values were between 2 and 20 microM for the preferred acyl-CoA substrates, and the Km values for L-carnitine varied depending on the acyl-CoA cosubstrates used. The Hill coefficient, n, was approximately 1 for all acyl-CoAs tested, indicating Michaelis-Menten kinetics. Carnitine octanoyltransferase retained its maximum activity when preincubated with 5,5'-dithiobis-(2-nitrobenzoate) at pH 7.0 or 8.5. Neither carnitine octanoyltransferase nor carnitine acetyltransferase were inhibited by malonyl-CoA. The immunology of carnitine octanoyltransferase is discussed. These data indicate that peroxisomal carnitine octanoyltransferase and carnitine acetyltransferase function in vivo in the direction of acylcarnitine formation, and suggest that the concentration of L-carnitine could influence the specificity for different acyl-CoA substrates.