Proton translocating ATPase in lysosomal membrane ghosts. Evidence that alkaline Mg2+-ATPase acts as a proton pump.
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 (delta pH). 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 degrees C. Its activity was inhibited by dicyclohexylcarbodiimide, 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'-disulfonic acid, that inhibited the H+-pump but not the alkaline Mg2+-ATPase, are discussed.
D009327:4-Chloro-7-nitrobenzofurazan / Q000494:pharmacology; D000251:Adenosine Triphosphatases / Q000378:metabolism*; D000818:Animals; D017301:Ca(2+) Mg(2+)-ATPase; D005033:Ethylmaleimide / Q000494:pharmacology; D006863:Hydrogen-Ion Concentration; D007425:Intracellular Membranes / Q000201:enzymology*; D007700:Kinetics; D008099:Liver / Q000201:enzymology*; D008247:Lysosomes / Q000201:enzymology*; D008297:Male; D006180:Proton-Translocating ATPases / Q000378:metabolism*; D011794:Quercetin / Q000494:pharmacology; D051381:Rats; D011919:Rats, Inbred Strains; D013053:Spectrophotometry / Q000379:methods
D011794:Quercetin; D000251:Adenosine Triphosphatases; D017301:Ca(2+) Mg(2+)-ATPase; D006180:Proton-Translocating ATPases; D009327:4-Chloro-7-nitrobenzofurazan; D005033:Ethylmaleimide