鼠肝细胞溶酶体膜中性pH p-对硝基苯磷酸酶的电镜细胞化学研究

用电镜细胞化学方法研究了大白鼠肝细胞溶酶体膜的中性pHp—对硝基苯磷酸酶(p-nltrophenyl phosphatase)(p-NPPase)的活性。切片用野田等人报告的原法反应时,溶酶体膜和基质均有酶的活性。加入酸性磷酸酶(AcPase)的抑制剂氟化钠(NaF)后,基质的酶活性被抑制,膜上的则不受影响。相反,当孵育液中含有4,4′-diisothiocyanostilbene-2.2′-disulfonic acid,disodium salt(DIDS)或N—乙基顺丁稀二酰亚胺(N-ethylmaleimide)(NEM)时,溶酶体膜上的酶反应被抑制而基质反应不受影响。如将氟化钠或DIDS或氟化钠和N—乙基顺丁烯二酰亚胺同时加到孵育液中,则膜和基质的反应都消失。这些结果表明溶酶体膜上中性范围的p—NPPase活性可能代表了H~+—ATPase活性。

Electron microscopic cytochemical studies of neutral pH p-NPPase on lysosomal membrane in rat liver cells

The neutral pH p-nitrophenyl phosphatase activity (p-NPPase) on the lysosomal membrane in rat liver cells was demonstrated with electron microscopic cytochemi-cal method. The enzyme activity was observed on the lysosomal membrane surfaces and its matrices when the section were treated with the reaction of the original incubation method reported by Noda et al. When sodium fluoride ( an inhibitor of acid phosphatase ) was added to the incubation medium, the enzyme activity on lysosomal matrices was inhibited but the activity on membrane was not. Either 4 , 4-diisothiocyanostilbene-2, 2-disulfonic acid,disodium salt ( DIDS ) or N-ethylmal-eimide (NEM)in the incubation medium could inhibit the enzyme activity on the lysosomal membrane but not in the matrices. The enzyme activity of both lysosomal membranes and its matrices was inhibited when the medium contained NaF and DIDS, or NaF and NEM at the same time. These results suggest that lysosomal membrane p-NPPase activity at neutral pH may represent H+-ATPase activity.