Tompa P, Batke J, Ovadi J, Welch G R, Srere P A
J Biol Chem. 1987 May 5;262(13):6089-92.
Formation of a bienzyme complex of pig heart mitochondrial malate dehydrogenase and citrate synthase in a buffered system is demonstrated by means of a covalently attached fluorescent probe to citrate synthase. Assuming 1:1 stoichiometry of the enzymes in the complex, an apparent dissociation constant of 10(-6) M was calculated from fluorescence anisotropy measurements. The effect of various metabolites on the interaction was tested. NAD+, oxalacetate, citrate, ATP, and L(-)- or D(+)-malate had no effect on the association of the two enzymes, whereas alpha-ketoglutarate increased and NADH decreased it. The interaction of mitochondrial citrate synthase with cytosolic malate dehydrogenase was found to be much weaker, whereas interaction of citrate synthase with another cytosolic enzyme, aldolase, could not be detected. In kinetic experiments, the activation of malate dehydrogenase by citrate synthase was observed. The effect of pyridine nucleotides and alpha-ketoglutarate is discussed in relation to the direction of the metabolic flow of oxalacetate.
通过将共价连接的荧光探针连接到柠檬酸合酶上，证明了在缓冲体系中猪心脏线粒体苹果酸脱氢酶和柠檬酸合酶形成了双酶复合物。假设复合物中酶的化学计量比为1:1，根据荧光各向异性测量计算出表观解离常数为10(-6) M。测试了各种代谢物对这种相互作用的影响。NAD+、草酰乙酸、柠檬酸、ATP以及L(-)-或D(+)-苹果酸对这两种酶的结合没有影响，而α-酮戊二酸增强了这种结合，NADH则减弱了这种结合。发现线粒体柠檬酸合酶与胞质苹果酸脱氢酶的相互作用要弱得多，而未检测到柠檬酸合酶与另一种胞质酶醛缩酶的相互作用。在动力学实验中，观察到柠檬酸合酶对苹果酸脱氢酶的激活作用。结合草酰乙酸代谢流的方向讨论了吡啶核苷酸和α-酮戊二酸的作用。
