Membrane ghosts were prepared from purified lysosomes (tritosomes) of rat liver by hypo-osmotic treatment. Mg
2+-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 Mg
2+-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 0.36mM 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 F
1F
0-ATPase in sensitivities to
N-ethylmaleimide, 7-chloro-4-nitrobenzo-2-oxa-1, 3-diazole, quercetin, and oligomycin.
Since this alkaline Mg
2+-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 Mg
2+-ATPase, are discussed.
抄録全体を表示