Yoshida H, Tonomura Y
J Biochem. 1976 Mar;79(3):649-54. doi: 10.1093/oxfordjournals.jbchem.a131109.
The time course of binding of N-ethylmaleimide (NEM) to the SR was measured at pH 7.5 in the presence or absence of ATP or ADP. The following results were obtained. 1. Both in the presence and absence of nucleotide, the ATPase [EC 3.6.1.3] activity decreased linearly with increase in the amount of NEM bound to the fragmented sarcoplasmic reticulum (SR), and was inhibited almost completely by the binding of 2 moles of NEM per 10(5) g of the SR protein. 2. The amount of NEM incorporated into the ATPase (M.W.=105,000) was measured by SDS disc-gel electrophoresis. It was shown that the ATPase activity was inhibited almost completely by the binding of 2 moles of NEM per mole of ATPase. 3. The rate of binding of NEM to SR decreased by 30-40% in the presence of either ATP or ADP. The concentrations of both ATP and ADP for half-saturation were 0.1-0.2mM. 4. The effect of nucleotide on the rate of binding of NEM was not changed by the presence of Ca2+ and Mg2+ ions. Similar effects were also observed even when the SR membranes were solubilized with Triton X-100. It is suggested from these results that one or two SH groups are located in the active site of the SR ATPase, and that conformational changes are induced by the addition of ATP and ADP.
在pH 7.5条件下，于有或无ATP或ADP存在时，测定了N - 乙基马来酰亚胺（NEM）与肌浆网（SR）结合的时间进程。得到以下结果。1. 无论有无核苷酸存在，ATP酶[EC 3.6.1.3]活性均随结合到破碎肌浆网（SR）上的NEM量增加而呈线性下降，并且每10(5) g SR蛋白结合2摩尔NEM时，ATP酶活性几乎完全被抑制。2. 通过SDS圆盘凝胶电泳测定了掺入ATP酶（分子量 = 105,000）中的NEM量。结果表明，每摩尔ATP酶结合2摩尔NEM时，ATP酶活性几乎完全被抑制。3. 在ATP或ADP存在时，NEM与SR的结合速率降低30 - 40%。半饱和时ATP和ADP的浓度均为0.1 - 0.2 mM。4. Ca2+和Mg2+离子的存在不改变核苷酸对NEM结合速率的影响。即使SR膜用Triton X - 100增溶，也观察到类似效果。从这些结果推测，SR ATP酶的活性位点有一个或两个SH基团，并且ATP和ADP的添加会诱导构象变化。
