Lawlis V B, Roche T E
Mol Cell Biochem. 1980 Nov 20;32(3):147-52. doi: 10.1007/BF00227441.
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.
在游离钙离子浓度为10微摩尔或无钙离子（即游离钙离子浓度低于1.0纳摩尔）的情况下，对牛肾α-酮戊二酸脱氢酶复合物的NADH抑制作用进行了比较。在有钙离子存在的情况下，对于广泛的NADH:NAD⁺比例，NADH抑制作用明显降低。当游离钙离子浓度略低于1微摩尔（用EGTA-Ca缓冲液测定）时，NADH抑制作用降低至最大值的一半。这必然是在钙离子对α-酮戊二酸S0.5的影响之上观察到的，钙离子使α-酮戊二酸的S0.5降低，在相似浓度下有一半最大效应。在二氢硫辛酰胺脱氢酶组分的测定中（使用二氢硫辛酰胺作为底物）或牛肾丙酮酸脱氢酶复合物的测定中，未观察到钙离子对NADH抑制作用的影响。这表明α-酮戊二酸脱氢酶复合物催化的整体反应是引发钙离子对NADH抑制作用所必需的。在固定的α-酮戊二酸浓度（50微摩尔）下，去除钙离子使α-酮戊二酸脱氢酶复合物的活性降低了8.5倍（由于α-酮戊二酸的S0.5增加），并且在不同的NADH:NAD⁺比例存在下，使复合物的活性降低了50至100倍。还描述了磷酸势（ATP/ADPxPi）或磷酸势与NADH:NAD⁺比例组合的影响。讨论了线粒体内游离钙离子水平作为通常与线粒体能量状态耦合的信号放大器的可能性。
