Department of Agronomy, University of Illinois, 1102 S. Goodwin Avenue, Urbana, Illinois 61801.
Plant Physiol. 1990 Oct;94(2):696-703. doi: 10.1104/pp.94.2.696.
Incubation of the red beet (Beta vulgaris L.) plasma membrane H(+)-ATPase with micromolar concentrations of diethylpyrocarbonate (DEPC) resulted in inhibition of both ATP hydrolytic and proton pumping activity. Enzyme activity was restored when DEPC-modified protein was incubated with hydroxylamine, suggesting specific modification of histidine residues. Kinetic analyses of DEPC inhibition performed on both membrane-bound and solubilized enzyme preparations suggested the presence of at least one essential histidine moiety per active site. Inclusion of either ATP (substrate) or ADP (product and competitive inhibitor) in the modification medium reduced the amount of inhibition observed in the presence of DEPC. However, protection was not entirely effective in returning activity to noninhibited control values. These results suggest that the modified histidine does not reside directly in the ATP binding region of the enzyme, but is more likely involved in enzyme regulation through subtle conformational effects.
用微摩尔浓度的二乙基焦碳酸酯（DEPC）孵育红甜菜（Beta vulgaris L.）质膜 H(+)-ATP 酶，会抑制 ATP 水解和质子泵的活性。当 DEPC 修饰的蛋白质与羟胺孵育时，酶活性得到恢复，这表明组氨酸残基被特异性修饰。在膜结合和可溶酶制剂上进行的 DEPC 抑制的动力学分析表明，每个活性位点至少存在一个必需的组氨酸部分。在修饰介质中包含 ATP（底物）或 ADP（产物和竞争性抑制剂）可减少在 DEPC 存在下观察到的抑制量。然而，保护措施并不能完全有效地将活性恢复到非抑制对照值。这些结果表明，修饰的组氨酸并不直接位于酶的 ATP 结合区域，而是更可能通过微妙的构象效应参与酶的调节。
