Abstract
Ketoglutarate dehydrogenase has been isolated from pig heart as a soluble complex of enzymes, containing protein bound thiamin pyrophosphate, lipoic acid and FAD in stoichiometric amounts. The flavin constituent of the complex has been shown to be lipoyl dehydrogenase. The complex was resolved into two fractions, one colourless, the other yellow, by fractionation in the presence of 2.5 M urea. The colourless fraction, which contains all of the protein-bound thiamin pyrophosphate and lipoic acid of the complex, was found to catalyse the oxidation of ketoglutarate when ferricyaide was used as electron acceptor. The yellow fraction was shown to be identical with lipoyl dehydrogenase (diaphorase). Neither fraction alone was capable of carrying out DPN +-linked ketoglutarate oxidation, this activity was reconstituted on mixing the two fractions. From chemical analysis, and the stoichiometry of the reconstitution of DPN +-linked ketoglutarate oxidation, the unit molecular weight of the ketoglutarate dehydrogenase complex was calculated to be about 260,000.
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