Proton Translocating ATPase in Lysosomal Membrane Ghosts. Evidence that Alkaline Mg2+-ATPase Acts as a Proton Pump1

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Abstract
Membrane ghosts were prepared from purified lysosomes (tritosomes) of rat liver by hypo-osmotic treatment. Mg2+-ATP-driven acidification was observed in the membrane ghosts using acridine orange as a fluorescent probe of the transmembrane pH gradient (ΔpH). Its properties were the same as those of intact lysosomes reported previously (Ohkuma, S., Moriyama, Y., & Takano, T. (1982) Proc. Natl. Acad. Sci. U.S.79, 2758–2762; Moriyama, Y., Takano, T., & Ohkuma, S. (1982) J. Biochem. 92, 1333–1336). The H+-pump was found to be electrogenic with use of bis(3-phenyl-5-oxoisoxasol-4-yl)pentamethine oxonol as a fluorescent membrane potential probe. Alkaline Mg2+-ATPase activity was also identified on the membranes. It showed a pH maximum of pH 8.0–8.5, a Km value for ATP of 0.36 mm and a Vmax of 0.41 units/mg protein at 30°C. Its activity was inhibited by dicyclohexylcar-bodiimide, tri-n-butyltin, azide and ADP, but not by ouabain or vanadate. It differed from mitochondrial F1F0-ATPase in sensitivities to N-ethylmaleimide, 7-chloro-4-nitrobenzo-2-oxa-1,3-diazole, quercetin, and oligomycin. Since this alkaline Mg2+-ATPase activity is very similar to the H+-pump activity in its requirement for divalent cations, substrate specificity and sensitivities to various chemicals, it may act as a proton translocase (H+-pump). Possible mechanisms of action of some chemicals, such as 4-acetamide-4′-isothiocyanatostilbene-2,2′-disul-fonic acid, that inhibited the H+-pump but not the alkaline Mg2+-ATPase, are discussed.