铈在溶酶体膜H^+-ATP酶电镜细胞化学中的应用

A NEW CERIUM-BASED METHOD FOR THE ULTRACYTOCHEMICAL DEMONSTRATION OF ACTIVITY OF H^+-ATPASE ON THE MENBRANE OF LYSOSOME AT PHYSIOLOGICAL pH

应用铈作捕捉剂的电镜细胞化学方法(简称铈法),对大鼠肝细胞溶酶体膜H^-ATP酶的活性进行了定位研究。用铈法的标准孵育液孵育肝组织切片时,酶的活性反应主要见于肝细胞的溶酶体膜和基质中;当加入NaF时,溶酶体基质内的反应被抑制;当加入N-乙基顺丁烯二酰亚胺(NEM)时,溶酶体膜上的反应被抑制;当同时加入NaF和NEM或不加底物时,溶酶体膜和基质内的反应均被抑制。

This paper reports on our work aimed at cytoceimical localization of the H^+-ATP-ase on the lysosomal embrane of liver cells by the Cerium-based cytochemical method.The reaction products were found on the lysosomal embrane and in its matrices by us-ing the standard incubation medium of Cerium-based method with p-NPP as ubstrate.When an inhibitor of acid phosphatase (10mmol/L NaF) was added to the incubaticnmedium, the enzyme ctivity in the lysosomal matrices was inhibited, but the activity onthe lysosomal membrane still remained. 20mmol/L EM (N-ethylmaleimide) added inthe incubation medium could inhibit the enzyme activitiy on the membrane of ysosomal.The enzyme activity both on the lysosomal membrane and in the lysosomal matrice wasinhibited when he edium contained both NaF and NEM. These results suggest that the p-NPPase on the lysosomal membrane at eutral H represents H^+-ATPase activi-ty. The reaction products of Cerium phosphate was finer and more stable. onspecificdeposits and diffusion of the reaction products were less and the localization of H^+-AT-Pase was much etter than that obtained by the leadbased method. We would emphasizethat the Cerium-based method for detecting he H^+-ATPase activity in the physiologicalOH should be popularized.