PURIFICATION AND CHARACTERIZATION OF SHORT-CHAIN, MEDIUM-CHAIN, AND LONG-CHAIN ACYL-COA DEHYDROGENASES FROM RAT-LIVER MITOCHONDRIA - ISOLATION OF THE HOLOENZYMES AND APOENZYMES AND CONVERSION OF THE APOENZYME TO THE HOLOENZYME

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  • 1 January 1985
    • journal article
    • research article
    • Vol. 260  (2) , 1311-1325
Abstract
Short-chain, medium-chain and long-chain acyl-CoA dehydrogenases were purified to homogeneity from rat liver mitochondria by sequential chromatography on DEAE-Sephadex A-50, hydroxyapatite, Matrex Gel Blue A, agarose-hexane-CoA and Bio-Gel A-0.5 m. Molecular, immunological and catalytic properties of the pure acyl-CoA dehydrogenases were investigated. The native MW of these 3 enzymes were 160,000, 180,000 and 180,000, respectively. The subunit MW of the 3 enzymes were estimated to be 41,000, 45,000 and 45,000, respectively, indicating that these enzymes are each composed of 4 subunits of equal size. The FAD content was calculated to be 1 mol/mol of subunit. While FAD binding by short-chain acyl-CoA dehydrogenase was very tight, that by medium-chain acyl-CoA and long-chain acyl-CoA dehydrogenases was less tight. The medium- and long-chain acyl-CoA dehydrogenases were also purified to homogeneity as FAD-free apoenzymes. The apoenzymes were converted to the fully active holoenzymes by incubation with FAD. The 3 acyl-CoA dehydrogenases were immunologically distinct from each other, i.e., the antibodies raised against the individual enzymes were monospecific and did not cross-react with any other acyl-CoA dehydrogenases. These preparations of the 3 enzymes exhibited substrate specificities (as defined in .**GRAPHIC**. and .**GRAPHIC**. significantly more specific than those of the previous preparations isolated from other sources. The substrate specificities were assessed also by measuring the activities in mitochondrial sonicates after selectively precipitating each enzyme with their individual monospecific antibodies. Butyryl-CoA was almost exclusively dehydrogenated by short-chain acyl-CoA dehydrogenase while C6-C10 acyl-CoA were mainly dehydrogenated by medium-chain cyl-CoA dehydrogenase. C14-C22 acyl-CoA were exclusively dehydrogenated by long-chain acyl-CoA dehydrogenase. C24 acyl-CoA were not dehydrogenated by this enzyme. Lauroyl-CoA appeared to be jointly dehydrogenated by the latter 2 enzymes. Branched-chain acyl-CoA were not dehydrogenated by short-chain acyl-CoA dehydrogenase. In the presence of electron-transfer flavoprotein or phenazine methosulfate, 2-enoyl-CoA were identified as products from the corresponding enzyme/acyl-CoA reactions.