Transmembrane Carboxyl Residues Are Essential for Cation-dependent Function in the Gastric H,K-ATPase

Rabon, E.; Hoggatt, Michele; Smillie, Kent

doi:10.1074/jbc.271.50.32137

Cited by 11 publications

(18 citation statements)

References 50 publications

(20 reference statements)

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“…10 mol of ATP/mg/h was hydrolyzed in the presence of 20 mM KCl and 120 mol/mg/h in the presence of KCl and nigericin or in the presence of 100 mM NH 4 Cl. Accordingly, 90% or more of the vesicles were ion tight.…”

Section: Methodsmentioning

confidence: 99%

“…The ATPase activity in the presence of K ϩ plus nigericin or of 100 mM NH 4 Cl was considered to reflect the total ATPase activity (24), whereas the difference between the latter measurements and the K ϩ -stimulated ATPase in the absence of ionophore reflects the contribution of ion tight vesicles. NH 4 ϩ activates the ATPase by permeating as NH 3 and then forming NH 4 ϩ , which is then transported by the H,K-ATPase out of the vesicles as a K ϩ surrogate resulting in cycling of the enzyme without the formation of a proton gradient (25). P i released was measured by the method of Yoda and Hokin (26) and protein by the Lowry method (27).…”

Section: Methodsmentioning

confidence: 99%

“…In summary, the enzyme under acid transporting conditions was incubated with the drug, and samples were taken at time zero and various other time points for measurement of labeling and of enzyme activity. Inhibition of enzyme activity was obtained by comparing the phosphate present in the samples at any given time point with the phosphate released following the addition of ATP and NH 4 ϩ to the samples as described previously (13,24). Thus, vesicles were added to a solution containing 250 mM sucrose, 150 mM KCl, 5 mM Tris/HCl, pH 6.8, and 1 g/ml valinomycin at 37°C to a final protein concentration of 100 -200 g/ml.…”

Section: Methodsmentioning

confidence: 99%

“…An additional 2 mM MgATP was added to start the reaction at 37°C. ATPase activity was measured in a solution of 40 mM Tris/HCl, pH 7, 100 mM NH 4 Cl, and 250 mM sucrose for 15 min. The ATPase activity remaining at each successive time point was obtained from the additional P i released in the second incubation.…”

Section: Methodsmentioning

confidence: 99%

“…For example, DCCD 1 is a hydrophobic reagent that reacts with a carboxylic group in the membrane domain of either pump in a K protectable manner (3,4) and thereby interfering not only with ATPase activity but also with Rb occlusion. Fluorescein isothiocyanate reacts with a cytoplasmic lysine in these pumps, providing a fluorescent marker (5,6) for Na-and K-induced conformations.…”

mentioning

confidence: 99%

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Sites of Reaction of the Gastric H,K-ATPase with Extracytoplasmic Thiol Reagents

Besancon

Simon²,

Sachs

et al. 1997

Journal of Biological Chemistry

241

178

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The vesicular gastric H,K-ATPase catalyzes an electroneutral H for K exchange allowing acidification of the intravesicular space. There is a total of 28 cysteines present in the ␣ subunit of the gastric H,K-ATPase, of which 10 are found in the predicted transmembrane segments and their connecting loop, and 9 are present in the ␤ subunit, of which 6 are disulfide-linked. To determine which of these was accessible to extracytoplasmic attack, the enzyme was inhibited by four different sub- , under acid transporting conditions. All of these compounds are weak bases that accumulate in the acidic space generated by the pump and undergo an acid catalyzed rearrangement to a cationic sulfenamide, which forms disulfides with accessible cysteines. The relative rates of acid activation of these compounds corresponded to the relative rates of inhibition of ATPase activity and acid transport. Fragmentation of the enzyme by trypsin followed by SDS-polyacrylamide gel electrophoresis showed that omeprazole bound covalently to one of the two cysteines in the domains containing the fifth and sixth transmembrane segments and their extracytoplasmic loop and to cysteine 892 in the loop between the seventh and eighth transmembrane segments, but inhibition correlated with the reaction with cysteines in the fifth and sixth domain. Lansoprazole bound to the cysteines in these two domains as well as to cysteine 321 toward the extracytoplasmic end of the third transmembrane segments. Pantoprazole bound only to either cysteine 813 or 822 in the fifth and sixth transmembrane region. The inhibition of Rabeprazole correlated also with its binding to this part of the protein, but this compound continued to bind after full inhibition, eventually binding also to cysteines 321 and 892. No binding was found to any of the cysteines in the seventh to tenth transmembrane segments. Thermolysin digestion of the isolated omeprazole-labeled fifth and sixth transmembrane pair showed that cysteine 813 was the site of labeling. It is concluded that binding of these sided reagents to cysteine 813 in the loop between transmembrane (TM)5 and TM6 is sufficient for inhibition of ATPase activity and acid transport by the gastric acid pump. Of the 10 cysteines present in the membrane and extracytoplasmic domain, only three are exposed sufficiently to allow reactivity with these cationic thiol reagents. The binding to cysteine 813 defines the location of the extracytoplasmic loop between TM5 and TM6 and places the carboxylic acids 820 and 824 conserved between the gastric H,K-and the Na,K-ATPases in TM6, consistent with their assumed role in cation binding.

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Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

mentioning

confidence: 99%

See 3 more Smart Citations

Sites of Reaction of the Gastric H,K-ATPase with Extracytoplasmic Thiol Reagents

Besancon

Simon²,

Sachs

et al. 1997

Journal of Biological Chemistry

241

178

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GastricH⁺,K⁺‐ATPase

Shin

Vagin

Munson

et al. 2008

Protein Science Encyclopedia

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Originally published in: Handbook of ATPase. Edited by Masamitsu Futai, Yoh Wada and Jack H. Kaplan. Copyright © 2004 Wiley‐VCH Verlag GmbH & Co. KGaA Weinheim. Print ISBN: 3‐527‐30689‐3 The sections in this article are Gastric H + , K + ‐ ATPase α Subunit of Gastric H + , K + ‐ ATPase β Subunit of Gastric H + , K + ‐ ATPase Regions of Association Between the α and β Subunits Regions of Association of the α Subunits Kinetics of the H + , K + ‐ ATPase Conformations of the H + , K + ‐ ATPase Functional Residues of the H + , K + ‐ ATPase Structural Model of the H + , K + ‐ ATPase Crystal Structure of the H + , K + ‐ ATPase Molecular Modeling of the Gastric H + , K + ‐ ATPase Acid Secretion and the H + , K + ‐ ATPase Inhibitors of the H + , K + ‐ ATPase Substituted Benzimidazoles Substituted Imidazo[1,2α]pyridines and other K + ‐competitive Antagonists Trafficking of the H + , K + ‐ ATPase

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Pepstatin A induces extracellular acidification distinct from aspartic protease inhibition in microglial cell lines

Okada

Irie

Sawada

et al. 2003

Glia

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The extrusion of protons is considered a very general parameter of the activation of many kinds of membrane or intracellular molecules, such as receptors, ion channels, and enzymes. We found that pepstatin A caused a reproducible, concentration-related increase in the extracellular acidification rate in two microglial cell lines, Ra2 and 6-3. Washing abolished pepstatin A-induced acidification immediately. However, pepstatin A did not cause the extracellular acidification in other cell types, such as CHO, C6 glioma, and NIH3T3 cells. These observations strongly suggest that pepstatin A interacts with certain membrane proteins specific to both Ra2 and 6-3 cells from outside. N-methylmaleimide and N,N'-dicyclohexylcarbodiimide, inhibitors of H(+)-ATPase, were found to reduce pepstatin A-induced response strongly, while bafilomycin A1, a vacuolar H(+)-ATPase inhibitor, vanadate, a P-type H(+)-ATPase inhibitor, and NaN3, an F1 ATPase inhibitor, virtually did not. 5-(N-ethyl-N-isopropyl) amiloride, an inhibitor of Na(+)/H(+) exchanger isoform 1, greatly enhanced pepstatin-induced response, while amiloride did not. Zn(2+), a voltage-dependent proton channel blocker, did not affect pepstatin-induced response neither. Staurosporine, a nonspecific inhibitor of protein kinase C, inhibited pepstatin A-induced response, while chelerythrine, more selective inhibitor of protein kinase C, greatly enhanced it. H-7 and H-8 did not affected the response. These findings suggest that pepstatin A induces extracellular acidification in microglia cell lines, Ra2 and 6-3, through an N-methylmaleimide- and N,N'-dicyclohexylcarbodiimide-sensitive, but bafilomycin A1-insensitive, ATPase, which seems to be distinct from protein kinase C-dependent process.

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Transmembrane Carboxyl Residues Are Essential for Cation-dependent Function in the Gastric H,K-ATPase

Cited by 11 publications

References 50 publications

Sites of Reaction of the Gastric H,K-ATPase with Extracytoplasmic Thiol Reagents

Sites of Reaction of the Gastric H,K-ATPase with Extracytoplasmic Thiol Reagents

GastricH⁺,K⁺‐ATPase

Pepstatin A induces extracellular acidification distinct from aspartic protease inhibition in microglial cell lines

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Transmembrane Carboxyl Residues Are Essential for Cation-dependent Function in the Gastric H,K-ATPase

Cited by 11 publications

References 50 publications

Sites of Reaction of the Gastric H,K-ATPase with Extracytoplasmic Thiol Reagents

Sites of Reaction of the Gastric H,K-ATPase with Extracytoplasmic Thiol Reagents

GastricH+,K+‐ATPase

Pepstatin A induces extracellular acidification distinct from aspartic protease inhibition in microglial cell lines

Contact Info

Product

Resources

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GastricH⁺,K⁺‐ATPase