Potassium-stimulated ATPase activity and hydrogen transport in gastric microsomal vesicles

Lee, Hon-Cheung; Breitbart, Haim; Berman, Marcia; Forte, John G.

doi:10.1016/0005-2736(79)90034-8

Cited by 85 publications

(40 citation statements)

References 37 publications

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“…Inorganic phosphate released was measured by the method of Fiske Subbarow (18). Previous studies on the hog gastric (H++ K+)-ATPase have suggested the optimal enzyme activity occurred around pH 7.0-7.5 (8,12,26). The pH optimum for the (H++K+) ATPase prepared by our method was 7.4, well-consistent with the above values.…”

Section: Methodssupporting

confidence: 88%

Purification and characterization of (H++K+)-ATPase from hog gastric mucosa.

1990

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Abstract-A (H++K+)-ATPase-enriched membrane fraction derived from the fundic portion of hog gastric mucosa was obtained by a combination of differential and repeated 7% Ficoll gradient centrifugation.The microsomal membrane fraction isolated by repeated 7% Ficoll gradient centrifugation was free of ouabain-sensitive (Na++K+)-ATPase, 5'-nucleotidase and succinate dehydrogenase; and it was highly enriched in (H++K+)-ATPase and K+-stimulated p-nitrophenylphosphatase (p-NPPase).The (H++K+)-ATPase had a pH optimum of 7.4 and was stimulated by TI', K+, Rb+ and NH4+ with Ka values of 0.0667, 0.526, 0.667 and 3.03 mM , respectively, at this pH. On the other hand, monovalent cations such as Na+ , Li+ and (CH3)4N+ as well as divalent cations such as Cue', Ca t+, Bat+, Sr2+ and Cd2+ inhibited this enzyme activity concentration-dependently.Ouabain and oligomycin had no effect, whereas omeprazole, a specific (H++K+)-ATPase inhibitor, inhibited this enzyme activity in a pH-dependent manner. Sodium dodecyl sulfate (SDS) polyacrylamide gel electrophoresis showed a major band (>90% of protein) at 97,400 daltons, which was phosphorylated in the presence of Mg2+ and [r-32P] ATP and dephosphorylated in the presence of K+. The present method was very simple, and the (H++K+)-ATPase activity of the microsomal fraction obtained by this method was much higher compared with those obtained by other methods such as free-flow electrophoresis.

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Section: Methodssupporting

confidence: 88%

Purification and characterization of (H++K+)-ATPase from hog gastric mucosa.

1990

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“…Protein A-agarose beads were from Pierce. Na 36 Cl and ACS II scintillant were obtained from GE Healthcare. 86 RbCl was from PerkinElmer Life Sciences.…”

Section: Methodsmentioning

confidence: 99%

“…Measurement of 36 Cl Ϫ Uptake into Hog Gastric Vesicles (SAV and TV)-SAV or TV (100 g of protein) were preincubated with a solution of 150 mM KCl, 250 mM sucrose, and 40 mM PIPES-Tris (pH 6.8) for 5 min or 20 h at 4°C. Then the vesicle (SAV or TV) was incubated with a solution containing 150 mM KCl, 4 mM MgSO 4 , 250 mM sucrose, 2 mM ATP, 5 Ci/ml Na 36 Cl, and 40 mM PIPES-Tris (pH 6.8) for 5 min at 25°C.…”

Section: Methodsmentioning

confidence: 99%

Functional Association between K+-Cl- Cotransporter-4 and H+,K+-ATPase in the Apical Canalicular Membrane of Gastric Parietal Cells

Fujii

Takahashi

Ikari

et al. 2009

Journal of Biological Chemistry

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We studied whether K ؉ -Cl ؊ cotransporters (KCCs) are involved in gastric HCl secretion. We found that KCC4 is expressed in the gastric parietal cells more abundantly at the luminal region of the gland than at the basal region. KCC4 was found in the stimulation-associated vesicles (SAV) derived from the apical canalicular membrane but not in the intracellular tubulovesicles, whereas H ؉ ,K ؉ -ATPase was expressed in both of them. In contrast, KCC1, KCC2, and KCC3 were not found in either SAV or tubulovesicles.

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“…The addition of valinomycin induced a secondary proton uptake due to increased K+ availability (5,12). The addition of TCS in the presence of valinomycin dissipated the proton gradient.…”

mentioning

confidence: 98%