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 language | English |
|---|---|
| Pages (from-to) | 995-1007 |
| Number of pages | 13 |
| Journal | Journal of Biochemistry |
| Volume | 95 |
| Issue number | 4 |
| Publication status | Published - Apr 1984 |
| Externally published | Yes |
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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 journal › Article
}
TY - JOUR
T1 - Proton translocating ATPase in lysosomal membrane ghosts. evidence that alkaline Mg-ATPase acts as a proton pump1
AU - Moriyama, Yoshinori
AU - Takano, Tatsuya
AU - Ohkuma, Shoji
PY - 1984/4
Y1 - 1984/4
N2 - 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.
AB - 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.
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M3 - Article
C2 - 6146604
AN - SCOPUS:0021369423
VL - 95
SP - 995
EP - 1007
JO - Journal of Biochemistry
JF - Journal of Biochemistry
SN - 0021-924X
IS - 4
ER -