Histochemical and cytochemical studies of neutral pH p-NPPase on lysosomal membrane in rat liver.

Abstract

The neutral pH p-nitrophenyl phosphatase activity (p-NPPase) on the lysosomal membrane in rat liver cells was demonstrated histo- and cytochemically. The enzyme activity was observed mainly on the lysosomal membrane surfaces and its matrices by using the original incubation medium consisting of 5 mM Mg salt p-NPP, 50 mM KCl, 2.5 mM levamisole, 25% DMSO and 2 mM lead citrate in 25 mM Tricine-KOH buffer, pH 7.3. When an inhibitor of acid phosphatase (5-10 mM, NaF) was added to the incubation medium, the enzyme activity on lysosomal matrices was inhibited but the activity on membranes was not. Either 4, 4-diisothiocyanatostilbene-2, 2′-disulphonic acid (10 mM DIDS) or N-ethylmaleimide (10 mM 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. Furthermore, if the Mg salt p-NPP was replaced by Na salt p-NPP in the NaF containing standard incubation medium, the enzyme activity was not observed at all on lysosomes. These results suggest that the lysosomal membrane p-NPPase activity at neutral pH may represent H+-ATPase activity.