Dubnova E B, Baykov A A
A.N. Belozersky Laboratory of Molecular Biology and Bioorganic Chemistry, Moscow State University, USSR.
Arch Biochem Biophys. 1992 Jan;292(1):16-9. doi: 10.1016/0003-9861(92)90044-w.
Intact rat liver mitochondria have very low hydrolytic activity, if any, toward exogenous pyrophosphate. The activity can be unmasked by making mitochondria permeable to PPi by toluene treatment or disrupting them with detergents or ultrasound, indicating that the active site of pyrophosphatase is located in the matrix. Initial rates of PPi hydrolysis by toluene-permeabilized mitochondria and purified pyrophosphatase were found to depend in a similar manner on PPi and Mg2+ concentrations. The simplest model consistent with the data in both cases implies that the reaction proceeds through two pathways and requires MgPPi as the substrate and, at least, one Mg2+ ion as the activator. In the presence of 0.4 mM Mg2+ (physiological concentration), the inhibition constant for Ca2+ is 12 microM and the enzyme activity is, at least, 50% maximal. The results suggest that the activity of pyrophosphatase in mitochondria is high enough to keep free PPi concentration at a level close to that at equilibrium.
完整的大鼠肝脏线粒体对外源性焦磷酸的水解活性非常低，即便有也极其微弱。通过甲苯处理使线粒体对焦磷酸具有通透性，或者用去污剂或超声破坏线粒体，这种活性就会显现出来，这表明焦磷酸酶的活性位点位于线粒体基质中。经发现，甲苯通透处理的线粒体和纯化的焦磷酸酶对焦磷酸的初始水解速率，以类似的方式取决于焦磷酸和镁离子的浓度。在这两种情况下，与数据相符的最简单模型表明，反应通过两条途径进行，需要MgPPi作为底物，并且至少需要一个镁离子作为激活剂。在存在0.4 mM镁离子（生理浓度）的情况下，钙离子的抑制常数为12 microM，且酶活性至少为最大值的50%。结果表明，线粒体中焦磷酸酶的活性足够高，能够将游离焦磷酸的浓度维持在接近平衡的水平。
