Cooperation of Ca2+ and pH in regulation of the activity of the 2-oxoglutarate dehydrogenase complex and its components from bovine kidney cortex.
A modified procedure for preparation of the 2-oxoglutarate dehydrogenase complex from bovine kidney cortex is presented. The enzymatic preparation obtained showed a specific activity of 18.5 mumol X min-1 X mg-1. This activity was dependent on Ca2+ (1-40 microM) and hydrogen ion concentration. At pH 7.6 in the absence of Ca2+ (less than 10(-9) M), S0.5 for 2-oxoglutarate was 2.5 mM, and in the presence of Ca2+ it was decreased to 0.3 mM. The maximum reaction rate at this pH was increased by Ca2+ by 33%. The increase of pH from 7.0 to 8.4 resulted in a 150-fold increase of S0.5. The activity of 2-oxoglutarate decarboxylase, a subunit of the dehydrogenase complex, was also dependent on Ca2+ and pH. The activity of 2-oxoglutarate decarboxylase, determined in the presence of ferrocyanide as electron acceptor, showed three different partial Michaelis constants for 2-oxoglutarate, low (K1m), medium (K2m) and high (K3m). At pH 6.9, K3m was 0.11 mM, and 0.005 mM in the absence and presence of Ca2+, respectively. The maximum reaction rate at pH 6.9 in the presence of Ca2+ was by 72% higher than in its absence. A change of pH from 6.9 to 7.6 led to an increase in K1m from 0.005 to 0.01 mM, and K3m from 0.11 to 0.60 mM. Ca2+ had no effect on the activity of lipoamide dehydrogenase or lipoamide succinyltransferase. These results indicate that, over the pH range 6.5 - 7.2, calcium ions affect the activity of the whole complex by regulating the activity of 2-oxoglutarate decarboxylase, whereas over the pH range 7.2 - 8.4 they affect the activity of the 2-oxoglutarate dehydrogenase complex by acting on the structure of the whole complex rather than by changing the activity of 2-oxoglutarate decarboxylase.
D000818:Animals; D002118:Calcium / Q000502:physiology*; D002417:Cattle; D002850:Chromatography, Gel; D008058:Dihydrolipoamide Dehydrogenase / Q000378:metabolism; D004533:Egtazic Acid / Q000494:pharmacology; D004591:Electrophoresis, Polyacrylamide Gel / Q000379:methods; D006863:Hydrogen-Ion Concentration; D066298:In Vitro Techniques; D007655:Ketoglutarate Dehydrogenase Complex / Q000378:metabolism*; D007658:Ketone Oxidoreductases / Q000378:metabolism*; D007672:Kidney Cortex / Q000201:enzymology*; D007700:Kinetics; D008928:Mitochondria / Q000201:enzymology; D011506:Proteins / Q000378:metabolism
D011506:Proteins; D004533:Egtazic Acid; D007658:Ketone Oxidoreductases; D007655:Ketoglutarate Dehydrogenase Complex; D008058:Dihydrolipoamide Dehydrogenase; D002118:Calcium