Cunningham C C, George D T
J Biol Chem. 1975 Mar 25;250(6):2036-44.
The lipid-free particulate preparations of the mitochondrial ATPase require phospholipid for activity and can be inhibited by oligomycin, as has been demonstrated previously. In this communication a steady state analysis of the activation of a particulate preparation of the ATPase by phospholipids and its subsequent inhibition by oligomycin has been carried out. The relative affinity of the ATPase for purified phospholipids has been determined by measuring the Km for activation (Ka) for several phospholipids. The Ka values varied from 30 to 100 mum. The Vmax in the presence of phosphatides varies from 0.29 to 1.11 mumol ATP hydrolyzed/min/mg of protein; no correlation is noted between the relative affinity of the enzyme for a phospholipid and the V max value. Higher V max values are noted with the more acidic phospholipids, however. Sodium dodecyl sulfate and monoolein also activate with Ka values of 25 and 800 mum, respectively. Diglycerides, however, do not activate. With all lipids the ATPase activity stimulated is oligomycin-sensitive. The Ki values for oligomycin range from 0.1 to 0.6 mum. Oligomycin is a competitive inhibitor with respect to all the phospholipids tested except phosphatidylethanolamine and phosphatidyglycerol. It is also competitive with respect to sodium dodecyl sulfate (k-i equals 0.94 mum). In reciprocal plots of activity versus ATP concentration, with and without oligomycin, an intercept consistent with either mixed or partial noncompetitive inhibition kinetics is noted. Comparable K-i values for oligomycin are obtained when calculated assuming either mixed or partial noncompetitive inhibition. The Km for ATP is the same in the unactivated and the lipid activated particulate ATPase; the value obtained is slightly lower than the Km for ATP in the solubilized, purified ATPase. Using a spectrophotometric assay the time required for activation with phospholipid and inhibition with oligomycin has also been determined. This investigation suggests the possibility that activation of the ATPase is due a position to interact with the water-soluble substrate. Consistent with the above suggestion is the supposition that the lipids do not necessarily confer inhibitor sensitivity to the ATPase, but rather allow an oligomycin-sensitive activity to be expressed.
线粒体ATP酶的无脂颗粒制剂需要磷脂来发挥活性，并且如先前所示，可被寡霉素抑制。在本通讯中，对磷脂激活ATP酶颗粒制剂及其随后被寡霉素抑制的过程进行了稳态分析。通过测量几种磷脂的激活米氏常数（Ka），确定了ATP酶对纯化磷脂的相对亲和力。Ka值在30至100 μM之间变化。在磷脂存在下的最大反应速度（Vmax）在0.29至1.11 μmol ATP水解/分钟/毫克蛋白质之间变化；未观察到酶对磷脂的相对亲和力与Vmax值之间存在相关性。然而，酸性更强的磷脂的Vmax值更高。十二烷基硫酸钠和单油酸甘油酯也能激活，其Ka值分别为25和800 μM。然而，甘油二酯不能激活。对于所有脂质，所刺激的ATP酶活性对寡霉素敏感。寡霉素的抑制常数（Ki）值在0.1至0.6 μM之间。除磷脂酰乙醇胺和磷脂酰甘油外，寡霉素对所有测试的磷脂都是竞争性抑制剂。它对十二烷基硫酸钠也是竞争性的（抑制常数ki等于0.94 μM）。在有和没有寡霉素的情况下，活性与ATP浓度的倒数图中，观察到与混合或部分非竞争性抑制动力学一致的截距。当假设为混合或部分非竞争性抑制进行计算时，可获得寡霉素类似的抑制常数。未激活的和脂质激活的颗粒ATP酶中ATP的米氏常数相同；所获得的值略低于溶解的、纯化的ATP酶中ATP的米氏常数。使用分光光度测定法还确定了用磷脂激活和用寡霉素抑制所需的时间。这项研究表明，ATP酶激活的可能性在于与水溶性底物相互作用的一个位置。与上述建议一致的假设是，脂质不一定赋予ATP酶抑制剂敏感性，而是允许表达对寡霉素敏感的活性。
