钙离子与猪心NAD⁺-异柠檬酸脱氢酶及2-氧代戊二酸脱氢酶复合物的结合

The binding of Ca2+ ions to pig heart NAD+-isocitrate dehydrogenase and the 2-oxoglutarate dehydrogenase complex.

Rutter G A, Denton R M

Department of Biochemistry, School of Medical Sciences, University of Bristol, U.K.

Biochem J. 1989 Oct 15;263(2):453-62. doi: 10.1042/bj2630453.

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.

通过流动透析和使用fura-2，研究了经小清蛋白/聚丙烯酰胺色谱法去除污染钙的纯化猪心NAD⁺-异柠檬酸脱氢酶和2-氧代戊二酸脱氢酶与Ca²⁺离子的结合情况。2. 对于2-氧代戊二酸脱氢酶复合物，每摩尔复合物有3.5摩尔的Ca²⁺结合位点，Ca²⁺的表观解离常数（Kd值）为2.0微摩尔。这些值受Mg²⁺离子、ADP或2-氧代戊二酸的影响很小。3. 相比之下，Ca²⁺与NAD⁺-异柠檬酸脱氢酶的结合（Kd = 14微摩尔）需要ADP、异柠檬酸和Mg²⁺离子。与NAD⁺-异柠檬酸脱氢酶相关的Ca²⁺结合位点数量为每摩尔四聚体酶0.9摩尔。4. 2-氧代戊二酸脱氢酶复合物将ADP（以ADP³⁻形式）结合到一组紧密结合位点（Kd = 3.1微摩尔），化学计量比为每摩尔复合物3.3摩尔，与Ca²⁺的结合情况相似；对于ADP³⁻，还存在数量可变的弱得多的位点（Kd = 100微摩尔）。

The binding of Ca2+ ions to pig heart NAD+-isocitrate dehydrogenase and the 2-oxoglutarate dehydrogenase complex.