Abstract
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.
Highlights
The 2-methyl branched-chain acyl-CoA dehydrogenase was purified thoomogeneity from mitochondria of the parasiticnematode, Ascaris mum
14.4 12.8 0 0 0 substituted for Medola blue in the spectrophotometric assay system. These resultssuggest that the ascarid electron-transport chain and, at least,two soluble mitochondrial proteins, electron-transfer flavoprotein (ETF) and 2-methyl branched-chainacyl-CoA dehydrogenase, are responsiblfeor thNe ADH-dependent reduction of branched-chain enoyl-CoAs observedin these organelles. In this studyw,e have purifiedthe 2-methyl branched-chain acyl-CoA dehydrogenase from A . s u u m mitochondria to homogeneityusinga series of rapid,stepwise elutions from DEAE-cellulose, Matrex Gel Blue A, andhydroxylapatite columns
It ipsossible that hydrogenase to function in its proposed physiological direc- the dehydrogenating activitoybserved with butyryl-CoA, valtion, as an enoyl-CoAreductase, the purifiedenzyme was eryl-CoA, and octanoyl-CoA results from the contamination incubated under nitrogen with NADHtig, lyl-CoA, purified A. of the purified 2-methyl branched-chain acyl-CoA dehydrosuum ETF, and freshly isolated A. s u m Submitochondrial particles (SMP), and 2-MeC4 genase with small amounts of short chain or medium chain formation was determined (TableVII)
Summary
The 2-methyl branched-chain acyl-CoA dehydrogenase was purified thoomogeneity from mitochondria of the parasiticnematode, Ascaris mum. Mol of subunit and the absorption coefficient of FAD oxidation found in mammalian mitochondria [7,8]. The purified enzyme dehydrogenated both 2-methylbutyryl-CoA and 2-methylvaleryl-CoA with apparent K,,a,nd V,, values of 18p~ and 1.62 pmol/min/mg and 21 p~ and 1.68 pmol/min/mg, respectively. This enzyme appeared to dehydrogenatebutyryl-CoA, valeryl-CoA, and octanoyl-CoA but at a much lower rate. Dehydrogendifferences should be anticipated since the ascarid enzymes function physiologicallyin the direction of acyl-CoA synthesis and not oxidation
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