Physicochemical Properties of the Diphosphopyridine Nucleotide-specific Isocitrate Dehydrogenase of Pig Heart

The DPN-dependent isocitrate dehydrogenase has been purified from pig heart by ion exchange chromatography and gel filtration. The resultant preparation exhibits a single polypeptide band, with a molecular weight of about 40,000, on polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate, suggesting that the enzyme is in a homogeneous state. The amino acid composition of this polypeptide chain is reported. In contrast, polyacrylamide disc gel electrophoresis of the enzyme preparation in the absence of denaturing reagents reveals a multiple protein pattern, with more than one enzymatically active species. The addition to the electrophoresis buffer of the substrate, isocitrate and manganous ion, or the allosteric activator, ADP, alters the protein pattern so that there is one major protein band. The multiple protein pattern observed in polyacrylamide disc gel electrophoresis thus appears to reflect different forms of the same enzyme. Isocitrate dehydrogenase becomes inactivated upon incubation at 4°; however, this cold inactivation can be reversed by incubation at 25° with isocitrate and manganous ion. Electrophoresis of the cold-inactivated preparations yields an increase in the protein species with faster mobility than the major enzyme band. It is proposed that at least part of the heterogeneity observed on electrophoresis of isocitrate dehydrogenase is attributable to cold inactivation occurring during the isolation procedures.