The binding of Ca2+ ions to pig heart NAD+-isocitrate dehydrogenase and the 2-oxoglutarate dehydrogenase complex.
1. The binding of Ca2+ ions to purified pig heart NAD+-isocitrate dehydrogenase and 2-oxoglutarate dehydrogenase, freed of contaminating Ca2+ by parvalbumin/polyacrylamide chromatography, has been studied by flow dialysis and by the use of fura-2. 2. For the 2-oxoglutarate dehydrogenase complex, 3.5 mol of Ca2+-binding sites/mol of complex were apparent, with an apparent dissociation constant (Kd value) for Ca2+ of 2.0 microM. These values were little affected by Mg2+ ions, ADP or 2-oxoglutarate. 3. By contrast, binding of Ca2+ to NAD+-isocitrate dehydrogenase (Kd = 14 microM) required ADP, isocitrate and Mg2+ ions. The number of Ca2+-binding sites associated with NAD+-isocitrate dehydrogenase was then 0.9 mol/mol of tetrameric enzyme. 4. The 2-oxoglutarate dehydrogenase complex bound ADP (as ADP3-) to a group of tight-binding sites (Kd = 3.1 microM) with a stoichiometry, 3.3 mol/mol of complex, similar to that for the binding of Ca2+; a variable number of much weaker sites (Kd = 100 microM) for ADP3- was also apparent.
Rutter
G A
GA
Department of Biochemistry, School of Medical Sciences, University of Bristol, U.K.
Denton
R M
RM
D000244:Adenosine Diphosphate / Q000378:metabolism / Q000494:pharmacology; D000818:Animals; D001572:Benzofurans; D001665:Binding Sites; D002118:Calcium / Q000378:metabolism*; D002147:Calmodulin / Q000378:metabolism; D002845:Chromatography; D003956:Dialysis; D005456:Fluorescent Dyes; D016257:Fura-2; D007521:Isocitrate Dehydrogenase / Q000378:metabolism*; D007655:Ketoglutarate Dehydrogenase Complex / Q000378:metabolism*; D007658:Ketone Oxidoreductases / Q000378:metabolism*; D008274:Magnesium / Q000494:pharmacology; D008929:Mitochondria, Heart / Q000201:enzymology*; D009243:NAD; D013552:Swine
D001572:Benzofurans; D002147:Calmodulin; D005456:Fluorescent Dyes; D009243:NAD; D000244:Adenosine Diphosphate; D007521:Isocitrate Dehydrogenase; D007658:Ketone Oxidoreductases; D007655:Ketoglutarate Dehydrogenase Complex; D008274:Magnesium; D002118:Calcium; D016257:Fura-2