Proton translocating ATPase in lysosomal membrane ghosts. evidence that alkaline Mg-ATPase acts as a proton pump1

Yoshinori Moriyama, Tatsuya Takano, Shoji Ohkuma

Research output: Contribution to journalArticle

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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.

Original languageEnglish
Pages (from-to)995-1007
Number of pages13
JournalJournal of Biochemistry
Volume95
Issue number4
Publication statusPublished - Apr 1984
Externally publishedYes

Fingerprint

Vacuolar Proton-Translocating ATPases
Proton Pumps
Proton-Translocating ATPases
ATP Synthase
Ca(2+) Mg(2+)-ATPase
Pump
Adenosine Triphosphatases
Protons
Membrane
Pumps
Membranes
Adenosinetriphosphate
Lysosomes
Probe
Adenosine Triphosphate
Pharmacologic Actions
Administrative data processing
Oligomycins
Acridine Orange
Membrane Potential

ASJC Scopus subject areas

  • Statistics, Probability and Uncertainty
  • Applied Mathematics
  • Physiology (medical)
  • Radiology Nuclear Medicine and imaging
  • Molecular Biology
  • Biochemistry

Cite this

Proton translocating ATPase in lysosomal membrane ghosts. evidence that alkaline Mg-ATPase acts as a proton pump1. / Moriyama, Yoshinori; Takano, Tatsuya; Ohkuma, Shoji.

In: Journal of Biochemistry, Vol. 95, No. 4, 04.1984, p. 995-1007.

Research output: Contribution to journalArticle

Moriyama, Yoshinori ; Takano, Tatsuya ; Ohkuma, Shoji. / Proton translocating ATPase in lysosomal membrane ghosts. evidence that alkaline Mg-ATPase acts as a proton pump1. In: Journal of Biochemistry. 1984 ; Vol. 95, No. 4. pp. 995-1007.
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