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.

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