Separation and properties of five distinct acyl-CoA dehydrogenases from rat liver mitochondria. Identification of a new 2-methyl branched chain acyl-CoA dehydrogenase.

Abstract

Five distinct acyl-CoA dehydrogenases were isolated from rat liver mitochondria using 3 5 4 0 % (NH&S04 precipitation, DEAE-Sephadex A-50 column chromatography, hydroxyapatite column chromatography, and isoelectric focusing.Sequential fractions obtained from these procedures were monitored for the various acyl-CoA dehydrogenase activities using three branched chain acyl-CoAs (isovaleryl-, 2-methylbutyryl-, and isobutyryl-CoA) and three straight chain acyl-CoAs with different chain lengths (n-butyryl-, noctanoyl-, and palmitoyl-CoA) as substrates.Two hitherto unknown dehydrogenases which are specific for branched chain acyl-CoAs were separated from the three previously known straight chain acyl-CoA dehydrogenases.These are 2-methyl branched chain acyl-CoA dehydrogenase and isovaleryl-CoA dehydrogenase.2-Methyl branched chain acyl-CoA dehydrogenase dehydrogenated 2-methylbutyryl-CoA and isobutyryl-CoA, but did not oxidize either isovaleryl-CoA or nbutyryl-CoA.Isovaleryl-CoA dehydrogenase dehydrogenated isovaleryl-CoA and n-valeryl-CoA but did not oxidize 2-methylbutyryl-CoA, isobutyryl-CoA, or n-butyryl-CoA at any significant rate.Short chain acyl-CoA, medium chain acyl-CoA, and long chain acyl-CoA dehydrogenases isolated in this manner, by close monitoring of the various acyl-CoA dehydrogenase activities in sequential fractions, had substrate specificities which were much narrower than those of the corresponding enzymes previously iso- lated.Using these isolated enzyme preparations and also crude preparations, effects of FAD addition and those of varying amounts of phenazinemethosulfate on activities of the individual enzymes were studied in order to establish the best assay conditions.The activity of all of the enzymes with an exception of short chain acyl-CoA dehydrogenase were enhanced 1.5-2.5-fold by 0.1 m M FAD addition.The affinity of FAD differed considerably from enzyme to enzyme.FAD in short chain acyl-CoA dehydrogenase was tightly bound and it was not lost during purification procedures.In contrast, medium chain acyl-CoA dehydrogenase readily lost FAD and its K , for FAD as measured by the enhancement of activity was 2.4 pM.The fractionation patterns for the five acyl-CoA dehydrogenases and properties of these five enzymes as presented here are useful as the basis for obtaining highly purified prepatutes of Health (AM 17453) and the March of Dimes (1-378).The