Malonyl-CoA decarboxylase from Mycobacterium tuberculosis and Pseudomonas fluorescens.

Malonyl-CoA decarboxylase was partially purified (nearly 1000-fold) from Mycobacterium tuberculosis H37Ra by ammonium sulfate precipitation, gel filtration with Sepharose 6B, and chromatography on DEAE Sephacel, carboxymethyl-Sephadex, and NADP-agarose. Polyacrylamide gel electrophoresis showed a major band (60–70%), which contained the enzymatic activity, and a minor band which had no decarboxylase activity. The molecular weight of the enzyme was 44,000, and the PI and pH optimum were 6.7 and 5.5, respectively. The enzyme showed a typical Michaelis-Menten substrate saturation, with an apparent K_m and V of 0.2 mm and 3.85 μmol/min/mg, respectively. It catalyzed decarboxylation of methylmalonyl-CoA only at 5% of the rate observed with malonyl-CoA, whereas malonic acid and succinyl-CoA were not decarboxylated. Antibodies prepared against malonyl-CoA decarboxylase from the uropygial glands of goose and rat liver mitochondria did not inhibit the bacterial enzyme. Avidin did not inhibit the enzyme suggesting that biotin was not involved in the reaction. Thiol-directed reagents inhibited the enzyme as did CoA, acetyl-CoA, propionyl-CoA, methylmalonyl-CoA, and succinyl-CoA. Malonyl-CoA decarboxylase was also partially purified from malonate-grown Pseudomonas fluorescens. The molecular weight of this enzyme was 56,000 and the pH optimum and apparent K_m were 5.5 and 1 mm, respectively. Unlike the mycobacterial enzyme, this enzyme was insensitive to p-hydroxymercuribenzoate, acetyl-CoA, and propionyl-CoA, and it was less sensitive to inhibition by succinyl-CoA and CoA than the mycobacterial enzyme. The size and properties of the two bacterial enzymes suggest that these are quite unlike the mammalian and avian enzymes and that they constitute a different class of malonyl-CoA decarboxylases.     

Malonyl-CoA Decarboxylase from Streptomyces erythreus: Purification,properties, and possible role in the production of erythromycin

Malonyl-CoA decarboxylase was purified (800-fold) from an erythromycin-producing strain of Streptomyces erythreus using DEAE-cellulose, Sephadex G-100, SP-Sephadex, and gel filtration with Sephadex G-75. The molecular weight of the native enzyme was 93,000 as determined by gel filtration and the subunit molecular weight was 45,000 as estimated by sodium dodecyl sulfate-polyacrylamide electrophoresis, suggesting an α₂ subunit composition for the native enzyme. Evidence is presented that during the purification procedure and storage a proteolytic cleavage occurred resulting in the formation of 30- and 15-kDa peptides. The enzyme showed a pH optimum of about 5.0 whereas the vertebrate enzyme showed an optimum at alkaline pH. The enzyme decarboxylated malonyl-CoA with a K_m of 143 μm and V of 250 nmol min^?1 mg^?1. For the decarboxylation of methylmalonyl-CoA this enzyme showed the opposite stereospecificity to that shown by vertebrate enzyme; the (R) isomer was decarboxylated at 3% of the rate observed with malonyl-CoA while the (S) isomer was not a substrate. Neither avidin nor biotin affected the rate of malonyl-CoA decarboxylation, suggesting that biotin is not involved in catalysis. Acetyl-CoA and free CoA were found to be competitive inhibitors. Propionyl-CoA, butyryl-CoA, succinyl-CoA, and methylmalonyl-CoA showed little inhibition, and neither thiol-directed reagents nor chelating agents inhibited the enzyme. High ionic strength and sulfate ions caused reversible inhibition of the enzymatic activity. Under two different cultural conditions the time course of appearance of malonyl-CoA decarboxylase was determined by measuring the enzyme activity and the level of the enzyme protein by an immunological method using rabbit antibodies prepared against the enzyme. In both cases the increase and decrease in the decarboxylase correlated with the rate of production of erythromycin, suggesting a possible role for this enzyme in the antibiotic production.     

Malonyl-CoA decarboxylase from the uropygial gland of waterfowl: purification, properties, immunological comparison, and role in regulating the synthesis of multimethyl-branched fatty acids.

Analysis of the acyl portion of the wax from the uropygial gland of muscovy duck, wood duck, (Cairininae subfamily) and Canadian goose (Anserinae) by combined gas-liquid chromatography and mass spectrometry showed that 2,4,6-trimethyloctanoic acid and 2,4,6-trimethylnonanoic acid were the major (~100%) components. Similar analyses of the wax from the glands of mallard and Peking duck (Anatinae) showed that 2- and 4-mono-methylhexanoic acids predominated (>75%) with no multimethyl-branched acids. The uropygial glands of the former group contained 20 to 100 times as much malonyl-CoA decarboxylase activity as those of the latter group. These results strongly support the hypothesis that this decarboxylase, by causing specific decarboxylation of malonyl-CoA, makes available only methylmalonyl-CoA for fatty acid synthesis, and thus causes the production of multimethyl-branched acids. Malonyl-CoA decarboxylase was purified to apparent homogeniety in 30% yield from the uropygial glands of muscovy and wood ducks. Properties of the enzyme from the ducks, such as S_20.w (7.8 S), molecular weight (190,000) subunit composition (4 × 47,000), amino acid composition, strict substrate specificity, pH optimum (~9.0), K_m (~33 μm), V (~80 μmol/min/mg), and inhibition by SH-directed reagents were similar to those observed with the decarboxylase from the domestic goose. Antiserum prepared against the goose enzyme cross-reacted with and inhibited the decarboxylase from the four genera of ducks and Canadian goose. Ouchterlony double-diffusion analyses showed fusion of precipitant lines with the enzyme from muscovy, wood duck, and Canadian goose, whereas spurs were observed with the enzymes from mallard and Peking ducks. Immunoelectrophoresis showed that the decarboxylases from muscovy and wood ducks were similar and that they were different from the enzyme from the domestic goose. It appears that during evolution, the subfamilies (Anserinae and Cairininae) which synthesize multimethyl-branched acids acquired the ability to produce a high level of malonyl-CoA decarboxylase, an enzyme which is also present in low levels in other organisms.     

Synthesis of multimethyl-branched fatty acids by avian and mammalian fatty acid synthetase and its regulation by malonyl-CoA decarboxylase in the uropygial gland

Fatty acid synthetase, partially purified by gel filtration with Sepharose 4B from goose liver, showed the same relative rate of incorporation of methylmalonyl-CoA (compared to malonyl-CoA) as that observed with the purified fatty acid synthetase from the uropygial gland. In the presence of acetyl-CoA, methylmalonyl-CoA was incorporated mainly into 2,4,6,8-tetramethyldecanoic acid and 2,4,6,8,10-pentamethyl-dodecanoic acid by the enzyme from both sources. Methylmalonyl-CoA was a competitive inhibitor with respect to malonyl-CoA for the enzyme from the gland just as previously observed for fatty acid synthetase from other animals. Furthermore, rabbit antiserum prepared against the gland enzyme cross-reacted with the liver enzyme, and Ouchterlony double-diffusion analyses showed complete fusion of the immunoprecipitant lines. The antiserum inhibited both the synthesis of n-fatty acids and branched fatty acids catalyzed by the synthetase from both liver and the uropygial gland. These results suggest that the synthetases from the two tissues are identical and that branched and n-fatty acids are synthesized by the same enzyme. Immunological examination of the 105,000g supernatant prepared from a variety of organs from the goose showed that only the uropygial gland contained a protein which cross-reacted with the antiserum prepared against malonyl-CoA decarboxylase purified from the gland. Thus, it is concluded that the reason for the synthesis of multimethyl-branched fatty acids by the fatty acid synthetase in the gland is that in this organ the tissue-specific and substrate-specific decarboxylase makes only methylmalonyl-CoA available to the synthetase. Fatty acid synthetase, partially purified from the mammary gland and the liver of rats, also catalyzed incorporation of [methyl-¹⁴C]methylmalonyl-CoA into 2,4,6,8-tetramethyldecanoic acid and 2,4,6,8-tetramethylundecanoic acid with acetyl-CoA and propionyl-CoA, respectively, as the primers. Evidence is also presented that fatty acids containing straight and branched regions can be generated by the fatty acid synthetase from the rat and goose, from methylmalonyl-CoA in the presence of malonyl-CoA or other precursors of n-fatty acids. These results provide support for the hypothesis that, under the pathological conditions which result in accumulation of methylmalonyl-CoA, abnormal branched acids can be generated by the fatty acid synthetase.     

Malonyl-CoA decarboxylase from Streptomyces erythreus: purification, properties, and possible role in the production of erythromycin

Malonyl-CoA decarboxylase was purified (800-fold) from an erythromycin-producing strain of Streptomyces erythreus using DEAE-cellulose, Sephadex G-100, SP-Sephadex, and gel filtration with Sephadex G-75. The molecular weight of the native enzyme was 93,000 as determined by gel filtration and the subunit molecular weight was 45,000 as estimated by sodium dodecyl sulfate-polyacrylamide electrophoresis, suggesting an alpha 2 subunit composition for the native enzyme. Evidence is presented that during the purification procedure and storage a proteolytic cleavage occurred resulting in the formation of 30- and 15-kDa peptides. The enzyme showed a pH optimum of about 5.0 whereas the vertebrate enzyme showed an optimum at alkaline pH. The enzyme decarboxylated malonyl-CoA with a Km of 143 microM and V of 250 nmol min-1 mg-1. For the decarboxylation of methylmalonyl-CoA this enzyme showed the opposite stereospecificity to that shown by vertebrate enzyme; the (R) isomer was decarboxylated at 3% of the rate observed with malonyl-CoA while the (S) isomer was not a substrate. Neither avidin nor biotin affected the rate of malonyl-CoA decarboxylation, suggesting that biotin is not involved in catalysis. Acetyl-CoA and free CoA were found to be competitive inhibitors. Propionyl-CoA, butyryl-CoA, succinyl-CoA, and methylmalonyl-CoA showed little inhibition, and neither thiol-directed reagents nor chelating agents inhibited the enzyme. High ionic strength and sulfate ions caused reversible inhibition of the enzymatic activity. Under two different cultural conditions the time course of appearance of malonyl-CoA decarboxylase was determined by measuring the enzyme activity and the level of enzyme protein by an immunological method using rabbit antibodies prepared against the enzyme. In both cases the increase and decrease in the decarboxylase correlated with the rate of production of erythromycin, suggesting a possible role for this enzyme in the antibiotic production.     

Precursor of a mitochondrial enzyme accumulates in the cytoplasm of preen glands for a specialized function

Malonyl-CoA decarboxylase in the mitochondria of the liver of goose is immunologically identical with the decarboxylase in the cytoplasm of the uropygial gland (Buckner et al. (1978) $\text{Arch. Biochem. Biophys.}$ 186, 152–163). Messenger RNA was isolated from the liver and the uropygial gland and translated in a rabbit reticulocyte system. Specific immunoprecipitation of the translation products with anti malonyl-CoA decarboxylase showed that in both cases the primary translation product was a 50 K dalton peptide identical in size to the cytoplasmic enzyme in the gland. Specific immunoprecipitation of malonyl-CoA decarboxylase from liver slices which had been incubated with [³⁵S]methionine showed that the mature mitochondrial enzyme was a 47 K dalton peptide, 3 K daltons smaller than the primary translation product and the isolated cytoplasmic enzyme. These results suggest that the decarboxylase is proteolytically processed during transport into the mitochondria and that the large amount of the cytoplasmic decarboxylase found in the gland represents accumulation of the unprocessed precursor form of the normally mitochondrial enzyme.     

Stereospecificity of malonyl-CoA decarboxylase, acetyl-CoA carboxylase, and fatty acid synthetase from the uropygial gland of goose

Malonyl-CoA decarboxylase from the uropygial gland of goose decarboxylated (R,S)-methylmalonyl-CoA at a slow rate and introduced 3H from [3H]2O into the resulting propionyl-CoA. Carboxylation of this labeled propionyl-CoA by propionyl-CoA carboxylase from pig heart and acetyl-CoA carboxylase from the uropygial gland completely removed 3H. Repeated treatment of (R,S)-[methyl-14C]methylmalonyl-CoA with the decarboxylase converted 50% of the substrate into propionyl-CoA, whereas (S)-methylmalonyl-CoA, generated by both carboxylases, was completely decarboxylated. Radioactive (R)- (S), and (R,S)-methylmalonyl-CoA were equally incorporated into fatty acids by fatty acid synthetase from the uropygial gland. The residual methylmalonyl-CoA remaining after fatty acid synthetase reaction on (R,S)-methylmalonyl-CoA was also racemic. These results show that: (a) the decarboxylase is stereospecific, (b) replacement of the carboxyl group by hydrogen occurs with retention of configuration, (c) acetyl-CoA carboxylase of the uropygial gland generates (S)-methylmalonyl-CoA from propionyl-CoA, and (d) fatty acid synthetase is not stereospecific for methylmalonyl-CoA.     

Inhibition in vitro of lipogenic enzymes from bovine (Bos taurus) mammary tissue by methylmalonyl-coenzyme A and coenzyme A

Bovine mammary fatty acid synthetase was inhibited by approximately 50% by 40 microM methylmalonyl-CoA; this inhibition was competitive with respect to malonyl-CoA (apparent Ki = 11 microM). Similarly, 6.25 microM coenzyme A inhibited the synthetase by 35% and this inhibition was again competitive (apparent Ki = 1.7 microM). Apparent Km for malonyl-CoA was 29 microM. The short-chain dicarboxylic acids malonic, methylmalonic and ethylmalonic at high concentrations (160-320 microM) and ATP (5 mM) enhanced the synthetase activity by about 50% respectively; the activating effects of methylmalonic acid and ATP on the synthetase were additive. Methylmalonyl-CoA at 50 microM concentration inhibited the partially purified acetyl-CoA carboxylase uncompetitively by 10% and the propionyl-CoA carboxylase activity of the enzyme preparation competitively (apparent Ki = 21 microM) by 40%. Malonyl-CoA also inhibited the acetyl-CoA carboxylase activity competitively (apparent Ki = 7 microM) by 35% and the propionyl-CoA carboxylating activity of the preparation competitively (apparent Ki = 4 microM) by 82%. The possibility that methylmalonyl-CoA may be a causal factor in the aetiology of the low milk-fat syndrome in high yielding dairy cows is discussed.     

Lipid biosynthesis in sebaceous glands: regulation of the synthesis of n- and branched fatty acids by malonyl-coenzyme A decarboxylase.

Crude cell-free extracts isolated from the uropygial glands of goose catalyzed the carboxylation of propionyl-CoA but not acetyl-CoA. However, a partially purified preparation catalyzed the carboxylation of both substrates and the characteristics of this carboxylase were similar to those reported for chicken liver carboxylase. The Km and Vmax for the carboxylation of either acetyl-CoA or propionyl-CoA were 1.5 times 10- minus-5 M and 0.8 mumol per min per mg, respectively. In the crude extracts an inhibitor of the acetyl-CoA carboxylase activity was detected. The inhibitor was partially purified and identified as a protein that catalyzed the rapid decarboxylation of malonyl-CoA. This enzyme was avidin-insenitive and highly specific for malonyl-CoA with very low rates of decarboxylation for methylmalonyl-CoA and malonic acid. Vmax and Km for malonyl-CoA decarboxylation, at the pH optimum of 9.5, were 12.5 mumol per min per mg and 8 times 10- minus-4 M, respectively. The relative activities of the acetyl-CoA carboxylase and malonyl-CoA decarboxylase were about 4 mumol per min per gland and 70 mumoles per min per gland, respectively. Therefore acetyl-CoA and methylmalonyl-CoA should be the major primer and elongating agent, respectively, present in the gland. The major fatty acid formed from these precursors by the fatty acid synthetase of the gland would be 2,4,6,8-tetramethyl-decanoic acid which is known to be the major fatty acid of the gland (Buckner, J. S. and Kolattukudy, P. E. (1975), Biochemistry, following paper). Therefore it is concluded that the malonyl-CoA decarboxylase controls fatty acid synthesis in this gland.     

Dual sites of occurrence of malonyl-CoA decarboxylase and their possible functional significance in avian tissues.

1. Uropygial glands of domestic goose and mallard which synthesize methyl-branched fatty acids, contain large quantities of cytosolic malonyl-CoA decarboxylase and a small quantity of mitochondrial enzyme. 2. Uropygial glands of chicken and the liver of geese which generate little methyl-branched acids, contain only small quantities of malonyl-CoA decarboxylase and in such cases the enzyme is in the mitochondria. 3. The mitochondrial decarboxylase from the uropygial gland and liver of goose is immunologically similar to the cytosolic decarboxylase of the uropygial gland. 4. The mitochondrial enzyme probably protects the mitochondrial enzymes which are susceptible to inhibition by malonyl-CoA, whereas the cytosolic enzyme promotes the synthesis of methyl-branched acids.     

Interaction of malonyl-CoA and related compounds with mitochondria from different rat tissues. Relationship between ligand binding and inhibition of carnitine palmitoyltransferase I.

The sensitivity of carnitine palmitoyltransferase I (CPT I; EC 2.3.1.21) to inhibition by malonyl-CoA and related compounds was examined in isolated mitochondria from liver, heart and skeletal muscle of the rat. In all three tissues the same order of inhibitory potency emerged: malonyl-CoA much greater than succinyl-CoA greater than methylmalonyl-CoA much greater than propionyl-CoA greater than acetyl-CoA. For any given agent, suppression of CPT I activity was much greater in skeletal muscle than in liver, with the heart enzyme having intermediate sensitivity. With skeletal-muscle mitochondria a high-affinity binding site for [14C]malonyl-CoA was readily demonstrable (Kd approx. 25 nM). The ability of other CoA esters to compete with [14C]malonyl-CoA for binding to the membrane paralleled their capacity to inhibit CPT I. Palmitoyl-CoA also competitively inhibited [14C]malonyl-CoA binding, in keeping with its known ability to overcome malonyl-CoA suppression of CPT I. For reasons not yet clear, free CoA displayed anomalous behaviour in that its competition for [14C]malonyl-CoA binding was disproportionately greater than its inhibition of CPT I. Three major conclusions are drawn. First, malonyl-CoA is not the only physiological compound capable of suppressing CPT I, since chemically related compounds, known to exist in cells, also share this property, particularly in tissues where the enzyme shows the greatest sensitivity to malonyl-CoA. Second, malonyl-CoA and its analogues appear to interact with the same site on the mitochondrial membrane, as may palmitoyl-CoA. Third, the degree of site occupancy by inhibitors governs the activity of CPT I.     

Molecular cloning, nucleotide sequence, and tissue distribution of malonyl-CoA decarboxylase

Malonyl-CoA decarboxylase was purified from goose uropygial gland, reduced, carboxymethylated, and digested with trypsin. Several peptides were purified by high performance liquid chromatography and their amino acid sequences determined. Oligonucleotide probes were prepared based on their amino acid sequences. Size-selected RNA from the goose uropygial gland was used to construct cDNA libraries in lambda gt11 and pUC9 vectors. Immunological screening of the lambda gt11 cDNA library yielded one clone, lambda DC1, which contained a 2.2-kilobase pair insert; hybridization with the synthetic oligonucleotide probes confirmed its identity as malonyl decarboxylase. Screening of the pUC9 cDNA library with the insert of lambda DC1 as a probe detected one clone, pDC2, with an insert of 2.9 kilobase pairs. The nucleotide sequences of the two cDNAs revealed an open reading frame encoding a polypeptide of 462 amino acids. The deduced amino acid sequence was confirmed as malonyl-CoA decarboxylase by matching it to the amino acid sequences of three tryptic peptides derived from mature enzyme. Northern blot analysis of mRNA from goose brain, kidney, liver, lung, and gland revealed malonyl-decarboxylase mRNA of 3000 nucleotides. Since clone pDC2 contains a 2928-nucleotide insert, it represents nearly the full length of mRNA. Brain, kidney, lung, and liver contained less than 1% of the malonyl-CoA decarboxylase mRNA in the gland. Southern blot analysis of genomic DNA showed a single band in both liver and gland, suggesting that malonyl-CoA decarboxylase is a single copy gene.     

Drosophila melanogaster fatty acid synthetase: Characteristics and effect of protease inhibitors

Fatty acid synthetase (FAS) from Drosophila melanogaster was purified by DEAE-cellulose and Sepharose CL-6B chromatography. Inclusion of protease inhibitors in all steps dramatically increased the specific activity of the FAS preparation (to an average value of 4500 U/mg protein, the highest value reported for any animal FAS). The relative molecular weight of the native enzyme was determined by gel filtration and found to be 480,000. SDS gel electrophoresis gave a subunit relative molecular weight of 226,000, indicating that D. melanogaster FAS, like other animal FASs, is a dimer. Acetyl-CoA was the most efficient primer with propionyl-CoA also supporting FAS activity. Neither hexanoyl-CoA butyryl-CoA, isobutyryl-CoA nor isovaleryl-CoA served as efficient primers. D. melanogaster FAS showed an absolute requirement for malonyl-CoA and no activity was observed when methylmalonyl-CoA replaced malonyl-CoA. However, in the presence of both elongating substrates, D. melanogaster FAS synthesized methyl branched fatty acids. Methylmalonyl-CoA appears to behave as a competitive inhibitor in the presence of malonyl-CoA     

A highly sensitive high-throughput luminescence assay for malonyl-CoA decarboxylase

Malonyl-CoA decarboxylase (MCD) catalyzes the conversion of malonyl-CoA to acetyl-CoA and thereby regulates malonyl-CoA levels in cells. Malonyl-CoA is a potent inhibitor of mitochondrial carnitine palmitoyltransferase-1, a key enzyme involved in the mitochondrial uptake of fatty acids for oxidation. Abnormally high rates of fatty acid oxidation contribute to ischemic damage. Inhibition of MCD leads to increased malonyl-CoA and therefore decreases fatty acid oxidation, representing a novel approach for the treatment of ischemic heart injury. The commonly used MCD assay monitors the production of NADH fluorometrically, which is not ideal for library screening due to potential fluorescent interference by certain compounds. Here we report a luminescence assay for MCD activity. This assay is less susceptible to fluorescent interference by compounds. Furthermore, it is 150-fold more sensitive, with a detection limit of 20 nM acetyl-CoA, compared to 3 μM in the fluorescence assay. This assay is also amenable to automation for high-throughput screening and yields excellent assay statistics (Z′ > 0.8). In addition, it can be applied to the screening for inhibitors of any other enzymes that generate acetyl-CoA.     

Ethylmalonyl-CoA decarboxylase, a new enzyme involved in metabolite proofreading

A limited number of enzymes are known that play a role analogous to DNA proofreading by eliminating non-classical metabolites formed by side activities of enzymes of intermediary metabolism. Because few such "metabolite proofreading enzymes" are known, our purpose was to search for an enzyme able to degrade ethylmalonyl-CoA, a potentially toxic metabolite formed at a low rate from butyryl-CoA by acetyl-CoA carboxylase and propionyl-CoA carboxylase, two major enzymes of lipid metabolism. We show that mammalian tissues contain a previously unknown enzyme that decarboxylates ethylmalonyl-CoA and, at lower rates, methylmalonyl-CoA but that does not act on malonyl-CoA. Ethylmalonyl-CoA decarboxylase is particularly abundant in brown adipose tissue, liver, and kidney in mice, and is essentially cytosolic. Because Escherichia coli methylmalonyl-CoA decarboxylase belongs to the family of enoyl-CoA hydratase (ECH), we searched mammalian databases for proteins of uncharacterized function belonging to the ECH family. Combining this database search approach with sequencing data obtained on a partially purified enzyme preparation, we identified ethylmalonyl-CoA decarboxylase as ECHDC1. We confirmed this identification by showing that recombinant mouse ECHDC1 has a substantial ethylmalonyl-CoA decarboxylase activity and a lower methylmalonyl-CoA decarboxylase activity but no malonyl-CoA decarboxylase or enoyl-CoA hydratase activity. Furthermore, ECHDC1-specific siRNAs decreased the ethylmalonyl-CoA decarboxylase activity in human cells and increased the formation of ethylmalonate, most particularly in cells incubated with butyrate. These findings indicate that ethylmalonyl-CoA decarboxylase may correct a side activity of acetyl-CoA carboxylase and suggest that its mutation may be involved in the development of certain forms of ethylmalonic aciduria.     

Radiochemical malonyl-CoA decarboxylase assay: activity and subcellular distribution in heart and skeletal muscle

Malonyl-CoA decarboxylase is the main route for the disposal of malonyl-CoA, the key metabolite in the regulation of mitochondrial fatty acid oxidation. We have developed a simple and sensitive radiochemical assay to determine malonyl-CoA decarboxylase activity. The decarboxylation of [2-14C]malonyl-CoA produces [2-14C]acetyl-CoA, which is converted to [2-14C]acetylcarnitine in the presence of excess L-carnitine and carnitine acetyltransferase. The positively charged radiolabeled product, acetylcarnitine, is separated from negatively charged excess radiolabeled substrate and the radioactivity measured by scintillation counting. Measurement of malonyl-CoA decarboxylase activities with this method gives values comparable to those obtained with assays currently in use, but has the advantage of being simpler and less labor intensive. We have applied this assay to rat skeletal muscle of different fiber-type composition and to rat heart. Malonyl-CoA decarboxylase activity (mU/g wet wt) correlates with the oxidative capacity of the muscles, being lowest in type IIb fibers (42.7 +/- 3.0) and highest in heart (1071.4 +/- 260), with intermediate activity in type IIa fibers (150.7 +/- 4.3) and type I fibers (107.8 +/- 7.6). Studies on subcellular distribution of malonyl-CoA decarboxylase activity in rat heart and rat skeletal muscle show that approximately 50 and 65% is localized to mitochondria, while 50 and 35% of the activity is extramitochondrial.     

Interacting effects of L-carnitine and malonyl-CoA on rat liver carnitine palmitoyltransferase.

Malonyl-CoA significantly increased the Km for L-carnitine of overt carnitine palmitoyltransferase in liver mitochondria from fed rats. This effect was observed when the molar palmitoyl-CoA/albumin concentration ratio was low (0.125-1.0), but not when it was higher (2.0). In the absence of malonyl-CoA, the Km for L-carnitine increased with increasing palmitoyl-CoA/albumin ratios. Malonyl-CoA did not increase the Km for L-carnitine in liver mitochondria from 24h-starved rats or in heart mitochondria from fed animals. The Km for L-carnitine of the latent form of carnitine palmitoyltransferase was 3-4 times that for the overt form of the enzyme. At low ratios of palmitoyl-CoA/albumin (0.5), the concentration of malonyl-CoA causing a 50% inhibition of overt carnitine palmitoyltransferase activity was decreased by 30% when assays with liver mitochondria from fed rats were performed at 100 microM-instead of 400 microM-carnitine. Such a decrease was not observed with liver mitochondria from starved animals. L-Carnitine displaced [14C]malonyl-CoA from liver mitochondrial binding sites. D-Carnitine was without effect. L-Carnitine did not displace [14C]malonyl-CoA from heart mitochondria. It is concluded that, under appropriate conditions, malonyl-CoA may decrease the effectiveness of L-carnitine as a substrate for the enzyme and that L-carnitine may decrease the effectiveness of malonyl-CoA to regulate the enzyme.     

Developmental changes in malonate-related enzymes of rat brain.

Mitochondria and high-speed supernatant were prepared from rat brain homogenates at 0–50 days of age. The development of malonyl-CoA synthetase, malonyl-CoA decarboxylase, coenzyme A-transferases and acetyl-CoA hydrolase was examined and compared to de novo fatty acid biosynthesis. The specific activity of malonyl-CoA synthetase rose steeply between 6 and 10 days, and this sudden increase coincided with peak specific activity of fatty acid synthetase. Similarly, malonate activation by coenzyme A-transfer from succinyl-CoA increased rapidly at the same time. Transfer of the coenzyme A moiety from acetoacetyl-CoA was only minimal during this period. Brain mitochondria had active malonyl-CoA decarboxylase which showed an almost linear increase of specific activity between 0 and 50 days. Acetyl-CoA resulting from malonyl-CoA decarboxylation underwent enzymatic hydrolysis to acetate and free coenzyme A. Only traces of acetoacetate were recovered. In mitochondria, acetyl-CoA hydrolase increased progressively whereas the cytosolic enzyme had high specific activity at birth which declined slowly during maturation.     

Interaction of malonyl-CoA and 2-tetradecylglycidyl-CoA with mitochondrial carnitine palmitoyltransferase I

Malonyl-CoA and 2-tetradecylglycidyl-CoA (TG-CoA) are potent inhibitors of mitochondrial carnitine palmitoyltransferase I (EC 2.3.1.21). To gain insight into their mode of action, the effects of both agents on mitochondria from rat liver and skeletal muscle were examined before and after membrane disruption with octylglucoside or digitonin. Pretreatment of intact mitochondria with TG-CoA caused almost total suppression of carnitine palmitoyltransferase I, with concomitant loss in malonyl-CoA binding capacity. However, subsequent membrane solubilization with octylglucoside resulted in high and equal carnitine palmitoyltransferase activity from control and TG-CoA pretreated mitochondria; neither solubilized preparation showed sensitivity to malonyl-CoA or TG-CoA. Upon removal of the detergent by dialysis the bulk of carnitine palmitoyltransferase was reincorporated into membrane vesicles, but the reinserted enzyme remained insensitive to both inhibitors. Carnitine palmitoyltransferase containing vesicles failed to bind malonyl-CoA. With increasing concentrations of digitonin, release of carnitine palmitoyltransferase paralleled disruption of the inner mitochondrial membrane, as reflected by the appearance of matrix enzymes in the soluble fraction. The profile of enzyme release was identical in control and TG-CoA pretreated mitochondria even though carnitine palmitoyltransferase I had been initially suppressed in the latter. Similar results were obtained when animals were treated with 2-tetradecylglycidate prior to the preparation of liver mitochondria. We conclude that malonyl-CoA and TG-CoA interact reversibly and irreversibly, respectively, with a common site on the mitochondrial (inner) membrane and that occupancy of this site causes inhibition of carnitine palmitoyltransferase I, but not of carnitine palmitoyltransferase II. Assuming that octylglucoside and digitonin do not selectively inactivate carnitine palmitoyltransferase I, the data suggest that both malonyl-CoA and TG-CoA interact with a regulatory locus that is closely juxtaposed to but distinct from the active site of the membrane-bound enzyme.