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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 hepatocyte lysosome membrane H + -ATPase was studied by using p-NPP as substrate and lead citrate as capture reagent. The results showed that When the standard incubation solution is incubated in hepatic tissue sections, the active product of the enzyme is mainly found in the lysosome membrane and matrix in hepatocytes; when sodium fluoride inhibitor (sodium fluoride), an inhibitor of acid phosphatase, is added, The reaction was inhibited and the reaction on the lysosomal membrane remained; when N-ethylmaleimide (NEM) was added, the reaction product on the lysosomal membrane was inhibited and the reaction within the matrix was still , While the addition of NaF and NEM at the same time, or the absence of substrate p-NPP in the standard incubation solution, the lysosomal membrane and matrix reaction were inhibited.The positive results suggested that the activity of p-NPP on lysosomal membrane Can represent the cytochemical localization of H + -ATPase; H + -ATPase on lysosomal membrane is a kind of H + -ATP that is different from mitochondrial F_0F_1-ATPase by Mg ~ (2 +), K + Enzymes.