大鼠肝细胞溶酶体膜H^+-ATP酶的组织细胞化学研究

HISTOCHEMICAL AND CYTOCHEMICAL STUDIES OF H^+-ATPase ON LYSOSOMAL MEMBRANE IN RAT LIVER

应用对硝基苯酚磷酸(p-NPP)做底物和柠檬酸铅做捕捉剂的孵育液,对大鼠肝细胞溶酶体膜H+-ATP酶进行了组织细胞化学的研究.结果表明,用标准孵育液孵育肝组织切片时,酶的活性反应产物主要见于肝细胞内的溶酶体膜和基质中;当加入酸性磷酸酶的抑制剂氟化钠(NaF)时,溶酶体基质中的反应被抑制,而溶酶体膜上的反应仍保留;当加入N-乙基顺丁烯二酰亚胺(NEM)时,溶酶体膜上的反应产物被抑制,而基质内的反应仍存在;当同时加入NaF和NEM,或标准孵育液中不加底物p-NPP时,溶酶体膜和基质内的反应均被抑制.阳性结果提示溶酶体膜上p-NPP酶的活性可代表H+-ATP酶的细胞化学定位;溶酶体膜上H+-ATP酶是由Mg^(2+),K+激活,被NEM抑制的,不同于线粒体F_0F_1-ATP酶的一种H+-ATP酶.

The H^+-ATPase activity on the lysosomal membrane in rat liver cells was demonstrated histo-cytochemically. The enzyme actiivity was observed mainly on the lysosomal membrane surfaces and its matrices by usiug the original incubation medium. When an inhibitor of acid phospatase was added to the incubation medium, the enzyme activity on lysosomal matrices was inhibited but the activity on membranes was not. N-ethylmaleimide added to incubation medium could inhibit the enzyme activity on the lysosomal membranes but not that in the matrices. The enzyme activity both on the lyosomal membranes and in its matrices was inhibited when the medium contained NaF and NEM at the same time. These results suggest that the lysosomal membrane p-NPPase activity at neutral pH may represent H^+-ATPase activity.