Marchesi V T, Palade G E
J Cell Biol. 1967 Nov;35(2):385-404. doi: 10.1083/jcb.35.2.385.
The lead salt method introduced by Wachstein and Meisel (12) for the cytochemical demonstration of ATPase activity was modified and used to determine sites of activity on red cell ghost membranes. Preliminary studies showed that aldehyde fixation and standard concentrations of the capture reagent Pb(NO(3))(2) resulted in marked inhibition of the ATPase activity of these membranes. By lowering the concentration of Pb(2+) and incubating unfixed red cell ghosts, over 50% of the total ATPase activity, which included an ouabain-sensitive, Na-K-activated component, could be demonstrated by quantitative biochemical assay. Cytochemical tests, carried out under the same conditions, gave a reaction product localized exclusively along the inner surfaces of the ghost membranes for both Mg-ATPase and Na-K-ATPase. These findings indicate that the ATPase activity of red cell ghosts results in the release of P(i) on the inside of the ghost membrane at sites scattered over its inner aspect. There were no deposits of reaction product on the outer surface of the ghost membrane, hence no indication that upon ATP hydrolysis P(i) is released outside the ghosts. Nor was there any clear difference in the localization of reaction product of Mg-ATPase as opposed to that of Na-K-ATPase.
由瓦施泰因（Wachstein）和迈泽尔（Meisel）（12）引入的用于细胞化学显示ATP酶活性的铅盐法经过改进，用于确定红细胞血影膜上的活性位点。初步研究表明，醛固定和捕获试剂硝酸铅（Pb(NO₃)₂）的标准浓度会导致这些膜的ATP酶活性受到显著抑制。通过降低Pb²⁺的浓度并孵育未固定的红细胞血影，通过定量生化分析可以证明超过50%的总ATP酶活性，其中包括一种对哇巴因敏感的、钠钾激活的成分。在相同条件下进行的细胞化学测试显示，镁ATP酶和钠钾ATP酶的反应产物仅定位在血影膜的内表面。这些发现表明，红细胞血影的ATP酶活性导致在血影膜内部其内侧分散的位点上释放无机磷酸（P(i)）。在血影膜的外表面没有反应产物沉积，因此没有迹象表明在ATP水解时P(i)会释放到血影外部。与钠钾ATP酶相比，镁ATP酶反应产物的定位也没有明显差异。
