Effect of micromolar Ca2+ on NADH inhibition of bovine kidney alpha-ketoglutarate dehydrogenase complex and possible role of Ca2+ in signal amplification.
NADH inhibition of bovine kidney alpha-ketoglutarate dehydrogenase complex was compared at 10 microM free Ca2+ or in the absence of Ca2+ (i.e., less than 1.0 nM free Ca2+). In the presence of Ca2+, NADH inhibition was appreciably decreased for a wide range of NADH:NAD+ ratios. A half-maximal decrease in NADH inhibition occurred at slightly less than 1 microM free Ca/+ (as determined with EGTA-Ca buffers). Of necessity this was observed on top of an effect of Ca2+ on the S0.5 for alpha-ketoglutarate which was decreased by Ca2+ with a half-maximal effect at a similar concentration. The effect of Ca2+ on NADH inhibition was not observed in assays of the dihydrolipoyl dehydrogenase component (using dihydrolipoamide as a substrate) or in assays of bovine kidney pyruvate dehydrogenase complex. This indicates that the overall reaction catalyzed by the alpha-ketoglutarate dehydrogenase complex is required to elicit the effect of Ca2+ on NADH inhibition. At a fixed alpha-ketoglutarate concentration (50 microM), removal of Ca2+ reduced the activity of the alpha-ketoglutarate dehydrogenase complex by 8.5-fold (due to an increase in S0.5 for alpha-ketoglutarate) and, in the presence of different NADH:NAD+ ratios, decreased the activity of the complex by 50 to 100-fold. Effects of the phosphate potential (ATP/ADPxPi) or a combination of the phosphate potential and NADH:NAD+ ratio are also described. The possibility that the level of intramitochondrial free Ca/+ serves as a signal amplifier normally coupled to the energy state of mitochondria is discussed.
D000818:Animals; D002118:Calcium / Q000494:pharmacology*; D002417:Cattle; D007655:Ketoglutarate Dehydrogenase Complex / Q000037:antagonists & inhibitors / Q000378:metabolism*; D007658:Ketone Oxidoreductases / Q000378:metabolism*; D007668:Kidney / Q000201:enzymology*; D008928:Mitochondria / Q000378:metabolism; D009243:NAD / Q000378:metabolism*
D009243:NAD; D007658:Ketone Oxidoreductases; D007655:Ketoglutarate Dehydrogenase Complex; D002118:Calcium