Glutamate dehydrogenase from peas: isolation, quaternary structure, and influence of cations on activity.
The most active multiple molecular form of glutamate dehydrogenase from pea seeds has been enriched 30000-fold with a recovery of 60--70%. The preparation is homogenous as judged from gel electrophoresis of native and dodecylsulfate-denatured enzyme and from analytical ultracentrifugation. Specific activities were 530 U/mg in the reductive amination and 90 U/mg in the oxidative deamination reaction. A sedimentation coefficient of s20, w = 10.49 S was determined. The specific volume and the molecular weight of the native enzyme were found to be v2 = 0.759 cm3/g and Mr = 260000, respectively, by equilibrium sedimentation in H2O and 90% 2H2O buffers. Dodecylsulfate electrophoresis of the denatured enzyme yielded a molecular weight of Mr = 44000 for the polypeptide chain. From our data we propose glutamate dehydrogenase from peas to be a hexamer. The hexameric structure is confirmed by the appearance of six electrophoretic bands after cross-linking with diimidates. Enzyme which was treated with Dowex A1 chelating resin was found to be almost completely inactive in the absence of divalent metal ions. From several metals tested, calcium was most efficient in reactivating the enzyme; half-maximal activity was attained at about 5 microM calcium. In contrast to potassium ions, sodium ions were found to interfere with this regulatory mechanism by activating the enzyme at high concentrations.
D000818:Animals; D002122:Calcium Chloride / Q000494:pharmacology; D002175:Candida / Q000201:enzymology; D002413:Cations, Divalent; D002417:Cattle; D003432:Cross-Linking Reagents; D007887:Fabaceae / Q000201:enzymology; D005969:Glutamate Dehydrogenase / Q000302:isolation & purification / Q000378:metabolism*; D046911:Macromolecular Substances; D008970:Molecular Weight; D010944:Plants / Q000201:enzymology*; D010946:Plants, Medicinal; D011188:Potassium / Q000494:pharmacology; D011489:Protein Denaturation; D012964:Sodium / Q000494:pharmacology
D002413:Cations, Divalent; D003432:Cross-Linking Reagents; D046911:Macromolecular Substances; D012964:Sodium; D005969:Glutamate Dehydrogenase; D002122:Calcium Chloride; D011188:Potassium