Molecular cloning of cDNA for rat liver general acyl CoA dehydrogenase and homology between the rat liver and pig kidney enzymes

cDNA clone for general acyl CoA dehydrogenase (GAD) was isolated from a rat liver cDNA expression library in lambda gt11 using anti-pig kidney GAD antibody. Size of the isolated cDNA was estimated to be 1.5-1.6 kb. By immunological analysis of fusion protein and epitope selection, the cDNA clone was identified as that containing the GAD gene. Partial amino acid sequence deduced from nucleotide sequence of the cDNA coincided with that of the pig kidney enzyme. The antibody cross-reacted with rat liver enzyme and molecular weights of these enzyme proteins were shown to be almost the same. All these results indicate that rat liver GAD shares a common structure with pig kidney enzyme.     

Acyl coenzyme A dehydrogenases and electron-transferring flavoprotein from beef hart mitochondria.

Electron-transferring flavoprotein (ETF) and long-chain acyl coenzyme A (CoA) dehydrogenase (LC-AD) have been purified essentially to homogeneity from beef heart (BH) mitochondria and partially characterized. The spectra of the major yellow acyl CoA dehydrogenase from BH mitochondria, both oxidized and when bleached with C₁₆CoA, were found to resemble those of pig liver (PL) LC-AD. The subunit molecular weight was found to be about 38,000 both by Na-dodecyl sulfate gel electrophoresis and by minimal molecular weight based on flavin content (A₄₅₀, ? = 11.3 × 10³ cm^?1m^?1). The enzyme is probably a tetramer with no interchain disulfide bonds. When assayed in the presence of ETF, relative activities are C₈CoA > C₁₆CoA ? C₄CoA. These findings show that physicochemical and specificity characteristics do not coincide in the pig liver and the beef heart enzymes. The BH ETF is similar to the PL ETF in its spectra, in subunit molecular weight determined by minimal molecular weight (based on flavin content as A₄₃₈), by Na-dodecyl-SO₄ gel electrophoresis, the absence of interchain disulfide bonds, V?_p, and the presence of two subunits/molecule. There were some changes in the amino acid composition concomitant with a decrease in apparent isoelectric point. The pig and beef enzymes were reactive with each other. The turnover number of the beef heart system at “saturating” ETF was 100 mol of 1, 6-dichlorophenol indophenol reduced/min/ mol of LC-AD. Abnormally low activity at low ETF concentrations as compared to high ETF concentrations was seen with the beef heart enzymes as with the pig liver system previously studied and again a material obtained during purification of the ETF similar to the “factor” from pig liver (based on chromatographie and disc-gel electrophoretic behavior) stimulated the low activity, while the high-ETF activity was relatively unaffected, permitting linear double-reciprocal plots over wide ranges of ETF concentration. Fatty-acid-free bovine serum albumin (BSA-FAF) could mimic this effect at equivalent protein concentrations (50–100 μg), as could increased LC-AD concentration and, to a lesser extent, limited aging. Studies of activity at very high concentrations of C₁₆CoA revealed a marked high-substrate inhibition with activity peaking at about 4 μm, the reported critical micelle concentration for C₁₆CoA. The addition of BSA-FAF resulted in more “normal” v vs [S] curves, and although the substrate inhibition was still present it was less severe. The K_m for C₁₆CoA in the presence of BSA-FAF is about 1 μm. These results suggest that the inhibitory species may be the C₁₆CoA micelle, and the BSA-FAF may reverse or alleviate the inhibition by binding acyl CoA in a manner analogous to its binding of fatty acid anions.     

Biosynthesis of porcine kidney D-amino acid oxidase

The biosynthesis of a porcine kidney peroxisomal enzyme, D-amino acid oxidase (EC 1.4.3.3., DAO), was investigated. Pig kidney mRNA as well as free and membrane-bound polysomes were used to investigate in vitro protein synthesis using a rabbit reticulocyte lysate. mRNA and free polysomes, but not membrane-bound polysomes, directed the synthesis of DAO. To examine the in vivo synthesis of the enzyme, a pig kidney cell line (LLC-PK1) was biosynthetically labelled. Both the in vitro and in vivo synthesized DAO had the same molecular weight, 38,000, as that of the purified enzyme. These results indicate strongly that DAO is synthesized on free ribosomes and transferred to the interior of peroxisomes without any proteolytic modification.     

Translation of messenger RNA of pig kidney D-amino acid oxidase in a cell-free system

In vitro synthesis of D-amino acid oxidase [D-amino acid: O2 oxidoreductase (deaminating), EC 1.4.3.3], one of the peroxisomal flavin enzymes, was performed using a rabbit reticulocyte lysate system in order to elucidate the biosynthetic pathway of the enzyme. The apparent molecular weight of the synthesized enzyme protein was the same as that of D-amino acid oxidase purified from pig kidney. On the other hand, the enzyme protein was not detectable when a wheat germ lysate system was used for the translation. Denaturation of pig kidney poly(A)+ RNA with methylmercury hydroxide prior to the translation was found to enhance the synthesis of the enzyme protein. These results suggest a tight conformational structure of the mRNA used.     

Genetics and ontogeny of butyryl CoA dehydrogenase in the mouse and linkage of Bcd-1 with Dao-1

A zymogram method has been developed for fatty acyl CoA dehydrogenase and used to examine the electrophoretic properties of butyryl CoA dehydrogenase (BCD) from mouse tissues. A single form of BCD is present in extracts of liver, kidney, heart, and intestine. Ontogenetic, tissue distribution, and subcellular fractionation results are consistent with the mitochondrial origin previously reported for this enzyme. A genetic variant for BCD-1 was used to provide evidence for a locus determining the electrophoretic properties of this enzyme (designated Bcd-1), which is linked to Dao-1 (encoding d-amino acid oxidase).This research was funded in part by the Australian Research Grants Committee.     

The subcellular distribution of acyl CoA: Dihydroxyacetone phosphate acyl transferase in guinea pig liver

Upon differential centrifugation, the enzyme acyl CoA: dihydroxyacetone phosphate acyl transferase (EC 2.3.1.42) in guinea pig liver is shown to sediment in a lysosomal-peroxisomal fraction. Comparison of the distribution of the marker enzymes and of DHAP acyl transferase indicates that the acyl transferase is localized in peroxisomes (microbodies).     

Partial purification and characterization of glutaryl-coenzyme A dehydrogenase, electron transfer flavoprotein, and electron transfer flavoprotein-Q oxidoreductase from Paracoccus denitrificans.

Glutaryl-coenzyme A (CoA) dehydrogenase and the electron transfer flavoprotein (ETF) of Paracoccus denitrificans were purified to homogeneity from cells grown with glutaric acid as the carbon source. Glutaryl-CoA dehydrogenase had a molecular weight of 180,000 and was made up of four identical subunits with molecular weights of about 43,000 each of which contained one flavin adenine dinucleotide molecule. The enzyme catalyzed an oxidative decarboxylation of glutaryl-CoA to crotonyl-CoA, was maximally stable at pH 5.0, and lost activity readily at pH values above 7.0. The enzyme had a pH optimum in the range of 8.0 to 8.5, a catalytic center activity of about 960 min-1, and apparent Michaelis constants for glutaryl-CoA and pig liver ETF of about 1.2 and 2.5 microM, respectively. P. denitrificans ETF had a visible spectrum identical to that of pig liver ETF and was made up of two subunits, only one of which contained a flavin adenine dinucleotide molecule. The isoelectric point of P. denitrificans ETF was 4.45 compared with 6.8 for pig liver ETF. P. denitrificans ETF accepted electrons not only from P. denitrificans glutaryl-CoA dehydrogenase, but also from the pig liver butyryl-CoA and octanoyl-CoA dehydrogenases. The apparent Vmax was of similar magnitude with either pig liver or P. denitrificans ETF as an electron acceptor for these dehydrogenases. P. denitrificans glutaryl-CoA dehydrogenase and ETF were used to assay for the reduction of ubiquinone 1 by ETF-Q oxidoreductase in cholate extracts of P. denitrificans membranes. The ETF-Q oxidoreductase from P. denitrificans could accept electrons from either the bacterial or the pig liver ETF. In either case, the apparent Km for ETF was infinitely high. P. denitrificans ETF-Q oxidoreductase was purified from contaminating paramagnets, and the resultant preparation had electron paramagnetic resonance signals at 2.081, 1.938, and 1.879 G, similar to those of the mitochondrial enzyme.     

Mitochondrial and peroxisomal beta-oxidations in pig coronary smooth muscle cells

1. The total subcellular membranes of pig coronary media were fractionated using a sucrose density gradient. 2. A fraction with high succinate dehydrogenase activity and a mean density of 1.165 was separated from a fraction with high catalase activity and a mean density of 1.145. 3. Acyl CoA beta-oxidation activity measured in the absence of BSA was present in both fractions with 47% of the total activity in the succinate dehydrogenase fraction and 47% in the catalase fraction. 4. In the succinate dehydrogenase fraction bovine serum albumin stimulated the acyl CoA beta-oxidation (maximal stimulation, 3.2 times at a concentration of 15 mg%) while in the catalase fraction it had no effect. 5. It is concluded that, in pig coronary media, the beta-oxidation system has two components, i.e. mitochondrial and peroxisomal beta-oxidation.     

Molecular weights of subunits of acetyl CoA carboxylase in rat liver cytoplasm

Monomeric [14C] methyl avidin was shown to bind to sodium dodecyl sulfate-denatured biotinyl proteins and remain bound through polyacrylamide gel electrophoresis which allowed their detection by fluorography. This method was used to show that purified rat liver acetyl CoA carboxylase contained two high molecular weight forms of the enzyme (MR = 241,000 and 252,000) while rapidly prepared, crude rat liver cytoplasm contained two larger molecular weight (MR = 257,000 and 270,000) forms. Thus, the enzyme had undergone substantial proteolysis during purification. The crude enzyme preparation also contained a smaller biotinyl protein (MR = 141,000) which is likely a proteolytic product of the larger forms of acetyl CoA carboxylase.     

A fluorescent assay to quantitatively measure in vitro acyl CoA:diacylglycerol acyltransferase activity

Triacylglycerols (TG) are the major storage form of energy in eukaryotic organisms and are synthesized primarily by acyl CoA:1,2-diacylglycerol acyltransferase (DGAT) enzymes. In vitro DGAT activity has previously been quantified by measuring the incorporation of either radiolabeled fatty acyl CoA or diacylglycerol (DG) into TG. We developed a modified acyltransferase assay using a fluorescent fatty acyl CoA substrate to accurately quantify in vitro DGAT activity. In the modified assay, radioactive fatty acyl CoA is replaced with fluorescent NBD-palmitoyl CoA, which is used as a substrate by DGAT with DG to produce NBD-TG. After extraction with organic solvents and separation by thin layer chromatography, NBD-TG formation can be detected and accurately quantified using a fluorescent imaging system. We demonstrate that this method can be adapted to detect other acyltransferase activities. Because NBD-palmitoyl CoA is commercially available at a much lower cost compared with radioactive acyl CoA substrates, it is a more economical alternative to radioactive tracers. In addition, the exposure of laboratory personnel to radioactivity is greatly reduced.     

Malate dehydrogenase: a higher molecular weight form produced by freeze-thaw treatment of pig heart supernatant enzyme.

Attempts to produce hybrids of pig heart supernatant and mitochondrial malate dehydrogenase with the use of guanidine hydrochloride, acid treatment, and freezethaw techniques have been unsuccessful. However, the freeze-thaw technique produced a catalytically active higher molecular weight form of supernatant malate dehydrogenase in the absence or presence of mitochondrial enzyme. The higher molecular weight of this artifact was established by gel filtration and gel electrophoresis criteria. The specific activity of the artifactual form of the enzyme appears to be close to that of native supernatant malate dehydrogenase.     

Coenzyme A- and NADH-dependent esterase activity of methylmalonate semialdehyde dehydrogenase.

Methylmalonate semialdehyde dehydrogenase purified to homogeneity from rat liver possesses, in addition to its coupled aldehyde dehydrogenase and CoA ester synthetic activity, the ability to hydrolyze p-nitrophenyl acetate. The following observations suggest that this activity is an active site phenomenon: (a) p-nitrophenyl acetate hydrolysis was inhibited by malonate semialdehyde, substrate for the dehydrogenase reaction; (b) p-nitrophenyl acetate was a strong competitive inhibitor of the dehydrogenase activity; (c) NAD+ and NADH activated the esterase activity; (d) coenzyme A, acceptor of acyl groups in the dehydrogenase reaction, accelerated the esterase activity; and (e) the product of the esterase reaction proceeding in the presence of coenzyme A was acetyl-CoA. These findings suggest that an S-acyl enzyme (thioester intermediate) is likely common to both the esterase reaction and the aldehyde dehydrogenase/CoA ester synthetic reaction.     

Purification and properties of an acyl CoA transferase from Ascaris suum muscle mitochondria

An acyl CoA transferase has been purified to electrophoretic homogeneity from the soluble compartment of Ascaris suum muscle mitochondria. From SDS-PAGE, isoelectric focusing and molecular exclusion chromatography, homogeneity was confirmed and the enzyme appears to be composed of two similar or identical subunits of apparent mol. wts of 50,000 resulting in an apparent mol. wt of 100,000 for the holoenzyme. The apparent isoelectric point was 5.6 +/- 0.1 by both chromatofocusing columns and slab gel isoelectric focusing. The transferase was relatively specific for the short, straight-chain acyl CoA donors as well as the CoA acceptors, being active on acetyl CoA, propionyl CoA, butyryl CoA, valeryl CoA and hexanoyl CoA as donors to acetate and propionate. Neither succinyl CoA nor succinate were appreciably active as CoA donor or acceptor, respectively. This enzyme cannot serve physiologically to activate succinate for decarboxylation to propionate, but may serve to ensure a supply of propionyl CoA which appears to be required in catalytic amounts for the decarboxylation of succinate.     

Target enzymes on hepatic dysfunction caused by dietary products of lipid peroxidation

Dietary products of lipid peroxidation cause hepatic dysfunction due to decreases in the activities of some hepatic enzymes and to depletion of CoA. An idea about the decreases and depletion is that the enzymes and CoA could be injured directly by the incorporated products in the liver. Their inactivations in vitro were then examined using a reasonable amount of peroxidation products. The hepatic cytosol, microsomes, and mitochondria were incubated with 10, 15, and 20 micrograms/mg protein of peroxidation products, respectively, and changes in the enzymatic activities were monitored. Glucose-6-phosphate dehydrogenase, mitochondrial NAD-dependent aldehyde dehydrogenase, glucokinase, and glyceradehyde phosphate dehydrogenase were inactivated, and the CoA level was decreased, but the other hepatic enzymes were not. Although glyceraldehyde phosphate dehydrogenase was most sensitive to peroxidation products in vitro, the decrease in activity was not detected by the oral dose of secondary products. On the other hand, among the components of peroxidation products, hydroperoxides and polymers are not incorporated in the liver, but decomposed products of low molecular weight are incorporated. Glucokinase among the above enzymes was not inactivated by the low-molecular-weight products. It was therefore concluded that glucose-6-phosphate dehydrogenase, mitochondrial NAD-dependent aldehyde dehydrogenase, and CoA were targets of the direct attack by incorporated components of peroxidation products in the liver.     

In vitro synthesis of pig pancreas ribonuclease

From studies on the in vitro synthesis of the heavily glycosylated pig pancreas ribonuclease (molecular weight of the protein moiety is 13 786, on the basis of the amino acid composition), the following points emerge: (1) the enzyme is synthesized as a precursor having an apparent molecular weight about 7000 higher than that of the mature non-glycosylated protein; (2) the mRNA coding for the enzyme protein consists of about 950 nucleotides.     

Selective screening for fatty acid oxidation disorders by tandem mass spectrometry: difficulties in practical discrimination

In a selective screening for fatty acid oxidation disorders by tandem mass spectrometry, we tested the diagnostic ratios and acylcarnitine concentrations in sera or blood spots, which were reported to be specific to very long-chain acyl CoA dehydrogenase deficiency, carnitine palmitoyltransferase I deficiency, and carnitine palmitoyltransferase II deficiency. While the acylcarnitine profiles in the majority of these patients were typical in the respective disorders, some overlapping of the indices was observed between these patients and the infants, who showed symptoms mainly related to hypoglycemia but did not have the disorders mentioned above. Although the diagnostic ratio of tetradecenoylcarnitine to dodecanoylcarnitine for very long-chain acyl CoA dehydrogenase deficiency seemed to minimize the overlapping in this study, additional measures including careful assessment of clinical data and enzyme assays may be necessary for the diagnosis in atypical cases.     

Purification and characterization of a cytoplasmic enzyme component of the Na+-activated malonate decarboxylase system of Malonomonas rubra: acetyl-S-acyl carrier protein: malonate acyl carrier protein-SH transferase

Malonate decarboxylation by crude extracts of Malonomonas rubra was specifically activated by Na⁺ and less efficiently by Li⁺ ions. The extracts contained an enzyme catalyzing CoA transfer from malonyl-CoA to acetate, yielding acetyl-CoA and malonate. After about a 26-fold purification of the malonyl-CoA:acetate CoA transferase, an almost pure enzyme was obtained, indicating that about 4% of the cellular protein consisted of the CoA transferase. This abundance of the transferase is in accord with its proposed role as an enzyme component of the malonate decarboxylase system, the key enzyme of energy metabolism in this organism. The apparent molecular weight of the polypeptide was 67,000 as revealed from SDS-polyacrylamide gel electrophoresis. A similar molecular weight was estimated for the native transferase by gel chromatography, indicating that the enzyme exists as a monomer. Kinetic analyses of the CoA transferase yielded the following: pH-optimum at pH 5.5, an apparent K_m for malonyl-CoA of 1.9mM, for acetate of 54mM, for acetyl-CoA of 6.9mM, and for malonate of 0.5mM. Malonate or citrate inhibited the enzyme with an apparent K_i of 0.4mM and 3.0mM, respectively. The isolated CoA transferase increased the activity of malonate decarboxylase of a crude enzyme system, in which part of the endogenous CoA transferase was inactivated by borohydride, about three-fold. These results indicate that the CoA transferase functions physiologically as a component of the malonate decarboxylase system, in which it catalyzes the transfer of acyl carrier protein from acetyl acyl carrier protein and malonate to yield malonyl acyl carrier protein and acetate. Malonate is thus activated on the enzyme by exchange for the catalytically important enzymebound acetyl thioester residues noted previously. This type of substrate activation resembles the catalytic mechanism of citrate lyase and citramalate lyase.Abbreviations DTNB 5,5 Dithiobis (2-nitrobenzoate) - MES 2-(N-Morpholino)ethanesulfonic acid - TAPS N-[Tris(hydroxymethyl)-methyl]-3-aminopropanesulfonic acid - SDS-PAGE sodium dodecyl sulfate-polyacrylamide gel electrophoresis     

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.     

Pig heart short chain L-3-hydroxyacyl-CoA dehydrogenase revisited: sequence analysis and crystal structure determination.

Short chain L-3-hydroxyacyl CoA dehydrogenase (SCHAD) is a soluble dimeric enzyme critical for oxidative metabolism of fatty acids. Its primary sequence has been reported to be conserved across numerous tissues and species with the notable exception of the pig heart homologue. Preliminary efforts to solve the crystal structure of the dimeric pig heart SCHAD suggested the unprecedented occurrence of three enzyme subunits within the asymmetric unit, a phenomenon that was thought to have hampered refinement of the initial chain tracing. The recently solved crystal coordinates of human heart SCHAD facilitated a molecular replacement solution to the pig heart SCHAD data. Refinement of the model, in conjunction with the nucleotide sequence for pig heart SCHAD determined in this paper, has demonstrated that the previously published pig heart SCHAD sequence was incorrect. Presented here are the corrected amino acid sequence and the high resolution crystal structure determined for pig heart SCHAD complexed with its NAD+ cofactor (2.8 A; R(cryst) = 22.4%, R(free) = 28.8%). In addition, the peculiar phenomenon of a dimeric enzyme crystallizing with three subunits contained in the asymmetric unit is described.     

Purification,properties, and kinetic mechanism of coenzyme A-linked aldehyde dehydrogenase from Clostridium kluyveri

Coenzyme A-linked aldehyde dehydrogenase from Clostridium kluyveri was purified from the soluble fraction of crude extracts and its physical and kinetic properties were studied. The enzyme was purified approximately 90-fold over crude extracts to a specific activity of 50 units/mg protein and was estimated to be 40% pure by polyacrylamide gel electrophoresis. From active enzyme centrifugation studies, aldehyde dehydrogenase was found to have a sedimentation coefficient of s_{20, w} = 7.4. The Stokes radius of the enzyme was determined by gel filtration and found to be 9.5 nm in the presence of substrates and 11.0 nm in the absence of substrates. Using the values found for the sedimentation coefficient and the Stokes radius, the molecular weight of the enzyme in the presence of substrates was calculated to be 290,000 and the frictional ratio, 2.2. Aldehyde dehydrogenase can utilize thiols other than CoA as acetyl acceptors. A number of methods were employed in order to exclude the possibility that these thiols act merely by recycling nonenzymatically trace amounts of CoA that might be in the enzyme preparation. From steady-state kinetic measurements, a ping pong mechanism was proposed in which NAD⁺ binds to free enzyme, acetaldehyde binds next, and NADH is released before CoA binds and acetyl-CoA released. At K_m levels of other substrates, substrate inhibition by CoA was observed. The nature of the substrate inhibition is discussed.