Lipid biosynthesis in the sebaceous glands: synthesis of multibranched fatty acids from methylmalonyl-coenzyme A in cell-free preparations from the uropygial gland of goose.

Cell-free extracts from the uropygial gland of goose catalyzed the incorporation of malonyl-CoA and methylmalonyl-CoA into n- and multi-branched fatty acids, respectively, with NADPH as the preferred reductant. Methylmalonyl-CoA was shown to be incorporated almost exclusively into the acyl portion of wax esters by the cell-free extract while malonyl-CoA was incorporated into polar lipids and both the acyl and alcohol portions of the wax. The optimal pH for the synthesis of both n- and multibranched acids was 6.0. Apparent Km and Vmax for malonyl-CoA were 2 times 10- minus-4 M and 250 nmol per min per mg, respectively, while the Km and Vmax for methylmalonyl-CoA were 7.7 times 10- minus-4 M and 0.8 nmol per min per mg, respectively with 105,000g supernatant; but partial purification resulted in a tenfold decrease in Km values. The partially purified synthetase preparation catalyzed the formation of n-C16 acid (80%) and n-C18 acid (20%) from acetyl-CoA and malonyl-CoA. With the same synthetase preparation and the appropriate primer methylmalonyl-CoA was converted into 2,4,6,8-tetramethyldecanoic acid and 2,4,6,8-tetramethylundecanoic acid which were identified by radio gas-liquid chromatography and combined gas chromatography-mass spectrometry. Experiments with an equimolecular mixture of acetyl-CoA and propionyl-CoA showed that the synthetase preferred acetyl-CoA as a primer. Since malonyl-CoA is known to be rapidly decarboxylated in the gland, acetyl-CoA and methylmalonyl-CoA are expected to be the major primer and elongating agent, respectively, available in the gland and therefore 2,4,6,8-tetramethyldecanoic acid should be the major product. Combined gas-liquid chromatography and mass spectrometry demonstrated that this acid was in fact the major acid of the gland.     

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.     

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.     

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.     

Precursor formation and biosynthesis of the macrolide antibiotic a 6599 (turimycin) by streptomyces hygroscopicus JA 6599]

The possible role of some metabolic systems producing acetyl-CoA, and methylmalonyl-CoA as initial precursors in the biosynthesis of the macrolide antibiotic A 6599 by Streptomyces hygroscopicus JA 6599 was studied. The activities of pyruvate decarboxylase exceeded in two higher producing strains about twofold those found in the mycelium of a lower producing one suggesting that in this organism an enhanced production of acetyl-CoA should be one of the prerequisites necessary for an improved antibiotic biosynthesis. No clear interrelationship was established, however, between the biosynthesis of the secondary metabolite A 6599 on the one hand and the acetate and propionate kinase content on the other hand. In S. hygroscopicus JA 6599 the carboxylation of acetyl-CoA or propionyl-CoA seems to be the major pathway giving malonyl-CoA or methylmalonyl-CoA, respectively. Thus, the activities of acetyl-CoA and propionyl-CoA carboxylases corresponded with both the levels of antibiotic production in several strains and with variations observed in the specific antibiotic production rate during the cultivation. Some other pathways synthesizing these precursors, e.g. via oxaloacetate, are assumed to be negligible since even in the mycelium of the lower producing strain increased activities of phosphoenolpyruvate carboxylase were present.     

Characterization of acetyl-CoA/propionyl-CoA carboxylase in Metallosphaera sedula. Carboxylating enzyme in the 3-hydroxypropionate cycle for autotrophic carbon fixation.

Autotrophic Archaea of the family Sulfolobaceae (Crenarchaeota) use a modified 3-hydroxypropionate cycle for carbon dioxide assimilation. In this cycle the ATP-dependent carboxylations of acetyl-CoA and propionyl-CoA to malonyl-CoA and methylmalonyl-CoA, respectively, represent the key CO2 fixation reactions. These reactions were studied in the thermophilic and acidophilic Metallosphaera sedula and are shown to be catalyzed by one single large enzyme, which acts equally well on acetyl-CoA and propionyl-CoA. The carboxylase was purified and characterized and the genes were cloned and sequenced. In contrast to the carboxylase of most other organisms, acetyl-CoA/propionyl-CoA carboxylase from M. sedula is active at 75 degrees C and is isolated as a stabile functional protein complex of 560 +/- 50 kDa. The enzyme consists of two large subunits of 57 kDa each representing biotin carboxylase (alpha) and carboxytransferase (gamma), respectively, and a small 18.6 kDa biotin carrier protein (beta). These subunits probably form an (alpha beta gamma)4 holoenzyme. It has a catalytic number of 28 s-1 at 65 degrees C and at the optimal pH of 7.5. The apparent Km values were 0.06 mm for acetyl-CoA, 0.07 mm for propionyl-CoA, 0.04 mm for ATP and 0.3 mm for bicarbonate. Acetyl-CoA/propionyl-CoA carboxylase is considered the main CO2 fixation enzyme of autotrophic members of Sulfolobaceae and the sequenced genomes of these Archaea contain the respective genes. Due to its stability the archaeal carboxylase may prove an ideal subject for further structural studies.     

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.     

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.     

Effects of manganese ions and magnesium ions on the activity of soya-bean ribulose bisphosphate carboxylase/oxygenase.

The Michaelis constants of soya-bean ribulose bisphosphate carboxylase for CO2 in the carboxylation reaction and for O2 in the oxygenation reaction depend on the nature of the bivalent cation present. In the presence of Mg2+ the Km for bicarbonate is 2.48 mM, and the Km for O2 is 37% (gas-phase concentration). With Mn2+ the values decrease to 0.85 mM and 1.7% respectively. For the carboxylation reaction Vmax. was 1.7 mumol/min per mg of protein with Mg2+ but only 0.29 mumol/min per mg of protein with Mn2+. For the oxygenation reaction, Vmax. values were 0.61 and 0.29 mumol/min per mg of protein respectively with Mg2+ and Mn2+.     

Propionyl-CoA carboxylase of Myxococcus xanthus: catalytic properties and function in developing cells

An acyl-coenzyme A carboxylase that carboxylates acetyl-CoA, butyryl-CoA, propionyl-CoA, and succinyl-CoA was purified from Myxococcus xanthus. Since the enzyme showed maximal rates of carboxylation with propionyl-CoA, the enzyme is thought to be propionyl-CoA carboxylase. The apparent K _m values for acetyl-CoA, butyryl-CoA, propionyl-CoA, and succinyl-CoA were found to be 0.2, 0.2, 0.03, and 1.0 mM, respectively. The native enzyme has a molecular mass of 605–615 kDa and is composed of nonidentical subunits (α and β) with molecular masses of 53 and 56 kDa, respectively. The enzyme showed maximal activity at pH 7.0–7.5 and at 25–30°C, and was affected by variation in concentrations of ATP and Mg²⁺. During development of M. xanthus, the propionyl-CoA carboxylase activity increased gradually, with maximum activity observed during the sporulation stage. Previous work has shown that a propionyl-CoA-carboxylase-deficient mutant of M. xanthus reduces levels of long-chain fatty acids. These results suggest that the propionyl-CoA carboxylase is also responsible for the carboxylation of acetyl-CoA to malonyl-CoA used for the synthesis of long-chain fatty acids during development. Received: 24 February 1998 / Accepted: 25 May 1998     

Regulation of malonyl-CoA concentration and turnover in the normal heart

The goal of this study was to test the relationship between malonyl-CoA concentration and its turnover measured in isolated rat hearts perfused with NaH(13)CO(3). This turnover is a direct measurement of the flux of acetyl-CoA carboxylation in the intact heart. It also reflects the rate of malonyl-CoA decarboxylation, i.e. the only known fate of malonyl-CoA in the heart. Conditions were selected to result in stable malonyl-CoA concentrations ranging from 1.5 to 5 nmol.g wet weight-(1). The malonyl-CoA concentration was directly correlated with the turnover of malonyl-CoA, ranging from 0.7 to 4.2 nmol.min(-) (1).g wet weight(-1) (slope = 0.98, r(2) = 0.94). The V(max) activities of acetyl-CoA carboxylase and of malonyl-CoA decarboxylase exceeded the rate of malonyl-CoA turnover by 2 orders of magnitude and did not correlate with either concentration or turnover of malonyl-CoA. However, conditions of perfusion that increased acetyl-CoA supply resulted in higher turnover and concentration, demonstrating that malonyl-CoA turnover is regulated by the supply of acetyl-CoA. The only condition where the activity of malonyl-CoA decarboxylase regulated malonyl-CoA kinetics was when the enzyme was pharmacologically inhibited, resulting in increased malonyl-CoA concentration and decreased turnover. Our data show that, in the absence of enzyme inhibitors, the rate of acetyl-CoA carboxylation is the main determinant of the malonyl-CoA concentration in the heart.     

Mammalian fatty acid synthetase. III. Characterization of human liver synthetase products and kinetics of methylmalonyl-CoA inhibition.

When propionyl-CoA was substituted for either acetyl-CoA or butyryl-CoA in the presence of [14C]malonyl-CoA and NADPH, the pure human liver fatty acids synthetase complex synthesized only straight-chain, saturated, 15- and 17-carbon radioactive fatty acids. At optimal concentrations, propionyl-CoA was a better primer of fatty acid synthesis than acetyl-CoA. Methylmalonyl-CoA inhibited the synthetase competitively with respect to malonyl-CoA. The Ki was calculated to be 8.4 muM. These findings provide an in vitro model and offer a direct explanation at the molecular level for some of the abnormal manifestations observed in diseases characterized by increased cellular concentrations of propionyl-CoA and methylmalonyl-CoA.     

The source of malonyl-CoA in rat heart. The calcium paradox releases acetyl-CoA carboxylase and not propionyl-CoA carboxylase

The formation of malonyl-CoA in rat heart is catalyzed by cytosolic acetyl-CoA carboxylase. The existence of this enzyme in heart is difficult to prove by the abundant occurrence of mitochondrial propionyl-CoA carboxylase, which is also able to catalyze the carboxylation of acetyl-CoA. We used the calcium paradox as a tool to separate cytosolic components from the remaining heart, and found that acetyl-CoA carboxylase activity was preferentially released, like lactate dehydrogenase and carnitine, while propionyl-CoA carboxylase was almost fully retained. Acetyl-CoA carboxylase activity was determined after activation by citrate ion and Mg2+. The activity decreased to 64% by 48 h of fasting.     

A role for hypothalamic malonyl-CoA in the control of food intake

The cellular level of malonyl-CoA, an intermediate in fatty acid biosynthesis, depends on its rate of synthesis catalyzed by acetyl-CoA carboxylase relative to its rate of utilization and degradation catalyzed by fatty acid synthase and malonyl-CoA decarboxylase, respectively. Recent evidence suggests that hypothalamic malonyl-CoA functions in the regulation of feeding behavior by altering the expression of key orexigenic and anorexigenic neuropeptides. Here we report that 5-aminoimidazole-4-carboxamide ribonucleoside (AICAR), a 5'-AMP kinase activator, rapidly lowers malonyl-CoA both in GT1-7 hypothalamic neurons and in the hypothalami of mice. These effects correlate closely with the phosphorylation of acetyl-CoA carboxylase, an established target of AMP kinase. Intracerebroventricular (i.c.v.) administration of AICAR rapidly lowers hypothalamic [malonyl-CoA] and increases food intake. Expression of an adenoviral cytosolic malonyl-CoA decarboxylase vector (Ad-cMCD) in hypothalamic GT1-7 cells decreases malonyl-CoA. When delivered by bilateral stereotaxic injection into the ventral hypothalamus (encompassing the arcuate nucleus) of mice, Ad-cMCD increases food intake and body weight. Ad-MCD delivered into the ventral hypothalamus also reverses the rapid suppression of food intake caused by i.c.v.-administered C75, a fatty acid synthase inhibitor that increases hypothalamic [malonyl-CoA]. Taken together these findings implicate malonyl-CoA in the hypothalamic regulation of feeding behavior.     

Stereochemistry of the methylmalonyl-CoA decarboxylation reaction

The steric course of the decarboxylation of (S)-methylmalonyl-CoA to propionyl-CoA, catalyzed by the biotin-dependent sodium pump methylmalonyl-CoA decarboxylase of Veillonella alcalescens was determined. The decarboxylation of (S)-methylmalonyl-CoA in 3H2O yielded (R)-[2-3H]propionyl-CoA; and the decarboxylation of (S)-[2-3H]methylmalonyl-CoA in H2O produced (S)-[2-3H]propionyl-CoA. The results demonstrate retention of configuration during the decarboxylation reaction. The substrate stereochemistry of methylmalonyl-CoA decarboxylase is thus the same as that of all other biotin-containing enzymes investigated.     

Purification and characterization of oxalyl-coenzyme A decarboxylase from Oxalobacter formigenes.

Oxalyl-coenzyme A (oxalyl-CoA) decarboxylase was purified from Oxalobacter formigenes by high-pressure liquid chromatography with hydrophobic interaction chromatography, DEAE anion-exchange chromatography, and gel permeation chromatography. The enzyme is made up of four identical subunits (Mr, 65,000) to give the active enzyme (Mr, 260,000). The enzyme catalyzed the thiamine PPi-dependent decarboxylation of oxalyl-CoA to formate and carbon dioxide. Apparent Km and Vmax values, respectively, were 0.24 mM and 0.25 mumol/min for oxalyl-CoA and 1.1 pM and 0.14 mumol/min for thiamine pyrophosphate. The maximum specific activity was 13.5 microM oxalyl-CoA decarboxylated per min per mg of protein.     

Decarboxylation of malonyl-CoA and biosynthesis of mevalonic acid in rat liver]

Biosynthesis of mevalonic acid (MVA), total formation of 14CO2 from [1,3-14C]malonyl-CoA and the activity of malonyl-CoA decarboxylase in subcellular fractions of rat liver were studied. The dependence of the rate of MVA biosynthesis on malonyl-CoA concentration was found to be linear both in 140,000 g supernatant and solubilized microsomal fractions. It was shown that in a composite system (140,000 g supernatant fraction added to washed microsomes, 10 : 1) the optimal concentration ratio for the substrates of MVA biosynthesis (malonyl-CoA and acetyl-CoA) is 1 to 2. In the absence of acetyl-CoA decarboxylation of [1,3-14C]malonyl-CoA was prevalent. In all subcellular fractions studied decarboxylation of [1,3-14C]malonyl-CoA prevailed over its incorporation into MVA, total non-saponified lipid fraction and fatty acids. The degree of malonyl-CoA, decarboxylation was not correlated with the rate of its incorporation into MVA, i. e. the increase in the 14CO2 formation was not accompanied by stimulation of [1,3-14C]malonyl-CoA incorporation either into MVA or into total non-saponified lipid fractions. The incorporation of [1-14C]acetyl-CoA into MVA under the same conditions was considerably lower than that of [1,3-14C]malonyl-CoA. In all subcellular fractions under study the activity of malonyl-CoA decarboxylase was found. The experimental data suggest that a remarkable part of malonyl-CoA is incorporated into MVA without preliminary decarboxylation. A possible role of malonyl-CoA decarboxylase as an enzyme which protects the cell against accumulation of malonyl-CoA and its immediate metabolites -- malonate and methylmalonyl-CoA is disucssed.     

Purification and characterization of a new sodium-transport decarboxylase. Methylmalonyl-CoA decarboxylase from Veillonella alcalescens

Upon resolution of the particulate cell fraction of Veillonella alcalescens by gel chromatography, membranes and ribosomes were clearly resolved. Methylmalonyl-CoA decarboxylase was bound to the membranes and not to ribosomes as reported earlier. Membrane vesicles containing methylmalonyl-CoA decarboxylase were prepared by disrupting V. alcalescens cells with a French pressure chamber. About 64% of the decarboxylase was oriented in these vesicles with the substrate binding site facing to the outside. The vesicles performed a rapid accumulation of Na+ ions in response to the decarboxylation of methylmalonyl-CoA. Decarboxylation and transport were highly uncoupled. The efficiency of the transport was considerably increased if methylmalonyl-CoA decarboxylation was retarded by using a low temperature or by slowly generating the substrate enzymically from propionyl-CoA. Under optimized conditions Na+ was concentrated inside the inverted vesicles eight-times higher than in the incubation medium. Methylmalonyl-CoA decarboxylase was solubilized from the membranes with Triton X-100 and purified about 20-fold by affinity chromatography on monomeric avidin-Sepharose columns. The decarboxylase was specifically activated by Na+ ions (apparent Km approximately equal to 0.6 mM). Whereas (S)-methylmalonyl-CoA was the superior substrate (apparent Km approximately equal to 7 microM), malonyl-CoA was also decarboxylated (apparent Km approximately equal to 35 microM). The decarboxylation of methylmalonyl-CoA yielded CO2 and not HCO-3 as the primary reaction product. Analysis of the purified enzyme by dodecylsulfate gel electrophoresis indicated the presence of four different polypeptides alpha, beta, gamma, delta with Mr 60 000, 33 000, 18 5000 and 14 000. The latter of these polypeptides was clearly visible only after silver staining but not after staining with Coomassie brilliant blue. A low molecular weight polypeptide with similar staining properties was also found in oxaloacetate decarboxylase. Methylmalonyl-CoA decarboxylase contained about 1 mol covalently bound biotin per 125 500 g protein which was localized exclusively in the gamma-subunit. This subunit therefore represents the biotin carboxyl carrier protein of methylmalonyl-CoA decarboxylase. A new very sensitive method for the detection of biotin-containing proteins is described.     

Purification and properties of malonyl-CoA decarboxylase from rat liver mitochondria and its immunological comparison with the enzymes from rat brain, heart, and mammary gland.

Malonyl-CoA decarboxylase (EC 4.1.1.9) was found to be localized in the mitochondria in rat liver. Low ionic strength (10 mm Na phosphate) buffer extracted the bulk (>85%) of the enzyme from the mitochondria. From this extract the enzyme was purified over 2,000-fold using a combination of (NH₄)₂SO₄ precipitation, gel filtration with Sepharose 4B and Sephadex G-150, ion exchange chromatography with QAE-Sephadex and CM-Sephadex, and finally chromatography on NADP-agarose. The purified enzyme, which had a specific activity of about 16 μmol/min/mg, appeared to be electrophoretically homogeneous and had a molecular weight of 160,000. The decarboxylase had a broad pH optimum between 8.5 and 10.0 and showed a typical Michaelis-Menten substrate saturation pattern from which K_m and V were calculated to be 54 μm and 18.8 μmol/min/mg, respectively. This enzyme decarboxylated neither malonic acid nor methylmalonyl-CoA and was severely inhibited by thiol-directed reagents such as p-hydroxymercuribenzoate and N-ethylmaleimide but not by iodoacetamide. Acetyl-CoA, propionyl-CoA, and methylmalonyl-CoA also inhibited the enzyme. The purified decarboxylase was immunogenic in rabbits and Ouchterlony double diffusion analysis revealed a single precipitant line with the purified enzyme. The IgG fraction isolated from the antiserum inhibited the enzyme from not only liver mitochondria but also the mammary gland, heart, and kidney of the rat. However, malonyl-CoA decarboxylase from rat brain mitochondria was not inhibited by the antibody. Malonyl-CoA decarboxylase purified from the uropygial gland of a domestic goose neither cross reacted nor was it inhibited by the antiserum prepared against the rat liver mitochondrial enzyme and the antibody against the goose enzyme neither cross-reacted nor inhibited the enzyme from the rat. It is proposed that a role for mitochondrial malonyl-CoA decarboxylase is to decarboxylate malonyl-CoA generated by propionyl-CoA carboxylase and thus protect mitochondrial enzymes susceptible to inhibition by malonyl-CoA.