Kinetic characterization of long chain fatty acyl coenzyme A ligase from rat liver mitochondria.

Long chain fatty acyl coenzyme A ligase (EC 6.2.1.3) purified from rat liver mitochondria has been characterized with respect to several kinetic parameters. Many of the kinetic properties of the mitochondrial enzyme are similar to those of the purified microsomal enzyme with respect to palmitoyl-CoA formation, but there are distinct differences. The fatty acid and nucleotide specificities of the mitochondrial enzyme are similar to those of the microsomal enzyme, as are the apparent Km values for ATP and coenzyme A. On the other hand, the mitochondrial enzyme differs from the microsomal enzyme in that it has a lower pH optimum, is different in molecular weight, and does not show simple saturation kinetics with palmitate as substrate. Of particular interest is the evidence presented which indicates that the mitochondrial long chain fatty acyl-CoA ligase, unlike short and medium chain ligases, does not utilize an acyladenylate as an intermediate in the formation of fatty acyl-CoA.     

Purification and characterization of aldehyde dehydrogenase from rat liver mitochondria

Nicotinamide adenine dinucleotide- and nicotinamide adenine dinucleotide phosphate-dependent dehydrogenase activities from rat liver mitochondria have been copurified to homogeneity using combined DEAE, Sepharose, and affinity chromatographic procedures. The enzyme has a native molecular weight of 240,000 and subunit molecular weight of 60,000. The enzyme is tetrameric consisting of four identical subunits as revealed by electrophoresis and terminal analyses. A partial summary of physical properties is provided. The amino acid composition by acid hydrolysis is reported. Specific activities for various NAD(P)+ analogs and alkanal substrates were compared. The action of the effectors chloral hydrate, disulfiram, diethylstilbestrol, and Mg2+ and K+ ions were also investigated.     

Purification and characterization of branched chain alpha-ketoacid dehydrogenase from bovine liver mitochondria.

Branched chain alpha-ketoacid dehydrogenase (EC 1.2.4.3(4)) was solubilized and purified from bovine liver mitochondria for the first time. Decarboxylation of alpha-ketoisovalerate, alpha-keto-beta-methylvalerate, and alpha-ketoisocaproate was catalyzed by this multienzyme complex and this activity was co-purified for each substrate. Three enzymatic functions were contained in the complex including decarboxylation of the above ketoacids, transacylation of their simple acid derivatives, and reduction of NAD+ as an overall reaction. Product stoichiometry of these three reactions was 1 CO2:1 acyl-CoA:1 NADH. Activity depended upon the addition of thiamin pyrophosphate, CoASH, and NAD+ which were dissociable cofactors. Physically, two active forms of the enzyme complex were found: a 275,000-dalton unit and a 2 x 10(6)-dalton component. Both showed a characteristic flavin spectra and catalyzed all functions of the complex, implying that 10 small units aggregated into the larger unit. The soluble complex as visualized by electron microscopy had a diameter ranging from 12 to 24 nm corresponding to a molecular weight of 2 x 10(6). The size of the native membrane-bound component remains to be determined.     

Dual coenzyme activities of high-Km aldehyde dehydrogenase from rat liver mitochondria

Various kinetic approaches were carried out to investigate kinetic attributes for the dual coenzyme activities of mitochondrial aldehyde dehydrogenase from rat liver. The enzyme catalyses NAD(+)- and NADP(+)-dependent oxidations of ethanal by an ordered bi-bi mechanism with NAD(P)+ as the first reactant bound and NAD(P)H as the last product released. The two coenzymes presumably interact with the kinetically identical site. NAD+ forms the dynamic binary complex with the enzyme, while the enzyme-NAD(P)H complex formation is associated with conformation change(s). A stopped-flow burst of NAD(P)H formation, followed by a slower steady-state turnover, suggests that either the deacylation or the release of NAD(P)H is rate limiting. Although NADP+ is reduced by a faster burst rate, NAD+ is slightly favored as the coenzyme by virtue of its marginally faster turnover rate.     

Purification and characterization of 2-oxoaldehyde dehydrogenase from rat liver.

The partial purification (123-fold) of 2-oxoaldehyde dehydrogenase (2-oxoaldehyde:NAD(P)+ oxidoreductase, 1.2.1.23) from rat liver was carried out using a purification procedure which involved (NH4)2SO4 fractionation, DEAE-Sephadex chromatography, Blue-Dextran affinity chromatography and CM-Sephadex chromatography. A single form of the enzyme was observed, mol. wt. approx. 50000 by gel chromatography. 2-Oxoaldehyde dehydrogenase appears to be highly specific for NADP+ and methylglyoxal. No activity is observed in the absence of certain amines which have vicinal amino and hydroxyl groups. The only known amine which activates the enzyme at physiological pH is L-serine methyl ester, suggesting that the regulation of this enzyme in vivo may require a derivative of serine.     

Purification and characterization of calmodulin from rat liver mitochondria

Mitochondrial calmodulin of rat liver was purified and classified. It co-migrated with bovine brain calmodulin in non-denaturing polyacrylamide gel electrophoresis, SDS-polyacrylamide gel electrophoresis and isoelectric focusing. The mitochondrial calmodulin activated Ca²⁺-dependent phosphodiesterase of bovine brain in the presence of Ca²⁺. About 80% of the mitochondrial calmodulin was proved to be of cytosol origin. It was easily detached by washing with buffer containing EGTA. The other 20% was intramitochondrial calmodulin; half of it was in the matrix space, and half in the membrane.     

Purification of L-3-glycerophosphate dehydrogenase from rat liver mitochondria

A rapid three-step procedure is presented for the purification of flavin-linked L-3-glycerophosphate dehydrogenase (E.C. 1.1.99.5.) from rat liver mitochondria. Solubilization of the enzyme is achieved selectively by digitonin, at a detergent-to-protein ratio of 0.7 mg/mg (mitochondrial protein concentration 10 mg/ml). The procedure involves chromatography on hydroxymethyl-hexamethylenediamine-succinyl-hexamethylenediamin e Sepharose 4B, followed by anion exchange chromatography using a FPLC technique. Subunit molecular weight of the enzyme was found to be 77,000 when prepared in the presence of the protease inhibitor phenylmethylsulphonyl fluoride. The Kmapp value for glycerophosphate was not influenced by the purification, and the ability of the enzyme to be activated by Ca2+ was preserved as well.     

Purification and properties of acyl coenzyme A dehydrogenases from bovine liver. Formation of 2-trans,4-cis-decadienoyl coenzyme A

Three straight chain acyl-CoA dehydrogenases were purified to apparent homogeneity from bovine liver using 40-70% (NH4)2SO4 precipitation, gel filtration, DEAE-cellulose column chromatography, and preparative electrophoresis. Separation of the acyl-CoA dehydrogenases by these procedures has been efficiently monitored by two newly developed analytical methods: (i) native staining of acyl-CoA dehydrogenases following separation by electrophoresis in polyacrylamide gels and (ii) determination of general acyl-CoA dehydrogenase by means of a specific substrate, 4-cis-decenoyl-CoA. The three acyl-CoA dehydrogenases were classified into short chain, general, and long chain acyl-CoA dehydrogenases on the basis of their chain length specificities according to the nomenclature proposed by Hall and Kamin (Hall, C. L., and Kamin, H. (1975) J. Biol. Chem. 250, 3470-3486). The enzymes gave single protein bands in polyacrylamide gel electrophoresis under denaturing and nondenaturing conditions, and their subunit and native molecular weights were estimated to be 40,300 and 188,000 for short chain acyl-CoA dehydrogenase, 43,300 and 205,000 for general acyl-CoA dehydrogenase, and 45,200 and 172,000 for long chain acyl-CoA dehydrogenase. Long chain and general acyl-CoA dehydrogenases markedly differed in their substrate specificities toward unsaturated acyl-CoA esters with a double bond at position 4. The former oxidized 4-cis-decenoyl-CoA at a rate of only 2.7% of that obtained with decanoyl-CoA as substrate, while for the latter enzyme 4-cis-decenoyl-CoA was even a slightly better substrate than decanoyl-CoA. 2-trans,4-cis-Decenoyl-CoA was identified as the product of this reaction.     

Purification and characterization of glycerol-3-phosphate dehydrogenase (flavin-linked) from rat liver mitochondria

We have purified the membrane-intrinsic glycerol-3-phosphate dehydrogenase from both normal and hyperthyroid rat liver mitochondria by extraction with Triton X-100, hydrophobic affinity chromatography, ion exchange chromatography, gel filtration, and FAD-linked Sepharose 4B affinity chromatography. The yields in both cases were over 20%, and purification ranged from 800- to 650-fold in mitochondria from hyperthyroid and normal rats, respectively. The final preparations appeared to be greater than 95% pure by polyacrylamide gel electrophoresis in the presence or absence of sodium dodecyl sulfate. The pure enzyme focused at pH 5.5 and produced a biphasic thermal inactivation plot at 50 degrees C. The holoenzyme was found to have a molecular mass of 250,000 daltons on gel filtration. The subunit molecular mass was found to be 74,000 daltons +/- 3,000 by sodium dodecyl sulfate-gel electrophoresis and high-performance liquid chromatography gel filtration in 0.1% sodium dodecyl sulfate. 1 mol of the holoenzyme preparation contains 1.1 mol of non-heme iron and 0.7-0.9 mol of noncovalently bound FAD. The absorption spectrum has a maximum at 375 nm and a shoulder at 450 nm which is bleached on treatment with sodium dithionite. The enzymatic reaction is competitively inhibited by glyceraldehyde 3-phosphate, dihydroxyacetone phosphate, phosphoenolpyruvate, and phosphoglycolic acid. The apparent Km for DL-alpha-glycerol 3-phosphate and noncovalently bound FAD were found to be 6 mM and 7 microM, respectively.     

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.     

Molecular cloning and nucleotide sequence of cDNA encoding the entire precursor of rat liver medium chain acyl coenzyme A dehydrogenase

cDNA encoding the precursor of rat liver medium chain acyl-CoA dehydrogenase (EC 1.3.99.3) was cloned and sequenced. The longest cDNA insert isolated was 1866 bases in length. This cDNA encodes the entire protein of 421-amino acids including a 25-amino acid leader peptide and a 396-amino acid mature polypeptide. The identity of the medium chain acyl-CoA dehydrogenase clone was confirmed by matching the amino acid sequence predicted from the cDNA to the NH2-terminal and nine internal tryptic peptide sequences derived from pure rat liver medium chain acyl-CoA dehydrogenase. The calculated molecular masses of the precursor medium chain acyl-CoA dehydrogenase, the mature medium chain acyl-CoA dehydrogenase, and the leader peptide are 46,600, 43,700, and 2,900 daltons, respectively. The leader peptide contains five basic amino acids and only one acidic amino acid; thus, it is positively charged, overall. Cysteine residues are unevenly distributed in the mature portion of the protein; five of six are found within the NH2-terminal half of the polypeptide. Comparison of medium chain acyl-CoA dehydrogenase sequence to other flavoproteins and enzymes which act on coenzyme A ester substrates did not lead to unambiguous identification of a possible FAD-binding site nor a coenzyme A-binding domain. The sequencing of other homologous acyl-CoA dehydrogenases will be informative in this regard.     

Purification of branched chain alpha-ketoacid dehydrogenase complex from rat liver

A new method using hydrophobic interaction chromatography on phenyl-Sepharose was developed to purify branched chain alpha-ketoacid dehydrogenase complex from commercially available frozen rat liver. Yields of greater than 50% were routinely achieved. The purified enzyme, composed of E1 alpha, E1 beta, and E2 subunits, appeared homogeneous on sodium dodecyl sulfate-polyacrylamide gel electrophoresis and contained endogenous kinase activity for phosphorylation and inactivation of the complex.