Quantitation of hydrogen ion and potential gradients in gastric plasma membrane vesicles

Rabon, E.; Chang, Hsuan H.; Sachs, George

doi:10.1021/bi00609a027

Cited by 87 publications

(38 citation statements)

References 26 publications

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“…In practice, this method has involved measuring alkalinization ofthe medium external to the vesicles, weakly buffered at pH 6.13. At this pH, the ATP hydrolytic reaction is isoprotic (1,4,27,29) so that any initial alkalinization of the medium should result from H+ transport into the vesicles. Measurement of this alkalinization has been conducted at relatively high levels of membrane protein using either a pH electrode (e.g.…”

Section: Resultsmentioning

confidence: 99%

“…In the kinetic approach, H+/ATP stoichiometry is estimated by a comparison of the rate of H+-translocation to the rate of ATP hydrolysis. Estimates of H+-translocation rate have been based upon either measuring external pH change in a weakly buffered medium using a pH electrode (4,29) or pH sensing dye (27), measuring the rate of H+ leakage from vesicles after the establishment of a steady-state pH gradient using a ApH sensing fluorescent probe (24) (26).…”

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confidence: 99%

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Determination of H⁺/ATP Stoichiometry for the Plasma Membrane H⁺-ATPase from Red Beet (Beta vulgaris L.) Storage Tissue

Briskin¹,

Niesman

1991

Plant Physiol.

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The H+/ATP stoichiometry was determined for the plasma membrane H+-ATPase from red beet (Beta vulgaris L., var Detroit Dark Red) storage tissue associated with native vesicles. The determination of H+/ATP stoichiometry utilized a kinetic approach where rates of H+ influx, estimated by three different methods, were compared to rates of ATP hydrolysis measured by the coupled enzyme assay under identical conditions. These methods for estimating H+ influx were based upon either determining the initial rate of alkalinization of the extemal medium from pH 6.13, measuring the rate of vesicle H+ leakage from a steadystate pH gradient after stopping the H+-ATPase or utilizing a mathematical model which describes the net transport of H+ at any given point in time. When the rate of H+ influx estimated by each of these methods was compared to the rate of ATP hydrolysis, a H+/ATP stoichiometry of about 1 was observed. In consideration of the maximum free energy available from ATP hydrolysis (AGATP), this value for H+/ATP stoichiometry is sufficient to account for the magnitude of the proton electrochemical gradient observed across the plasma membrane in vivo.Through in vitro studies with isolated plasma membrane vesicles (1 1, 13) and reconstituted enzyme systems (7,21,30,32), it has been demonstrated that the plasma membrane H+-ATPase has the capability of coupling ATP hydrolysis to vectorial H+-translocation. In vivo, it is proposed that this activity results in the production of a proton electrochemical gradient across the plasma membrane consisting of both an interior-negative membrane potential and acid-exterior pH difference (16,18,31 membrane H+-ATPase forms a covalent phosphorylated intermediate during the course of ATP hydrolysis (8, 9) on an essential aspartyl residue in the active site of the enzyme (10, 34). A further similarity to other transport ATPases of the EIE2 type regards the structure ofthe plant plasma membrane H+-ATPase where the functional unit of ATPase activity is associated with a 100 kD catalytic subunit (8, 9). Recent efforts by several research groups (5, 15, 22) have resulted in the successful cloning of the plasma membrane H+-ATPase gene and the deduction of the primary amino acid sequence for this protein. Of prime importance will be developing an understanding of structure/function relationships for the mechanism of energy coupling to H+-transport as mediated by this protein.An important parameter associated with the H+-transport function of the plasma membrane H+-ATPase is the stoichiometric relationship between the number of H+ transported and ATP molecules hydrolyzed. This H+/ATP value sets an upper limit to the magnitude of the proton electrochemical gradient that can be established across the plasma membrane (25, 28) and would be an important consideration in the development of mechanistic models to explain the catalytic/ transport function of this enzyme.In the determination of H+/ATP stoichiometry for an H+-ATPase, either a thermodynamic or kinetic approach could be utilized ...

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Section: Resultsmentioning

confidence: 99%

mentioning

confidence: 99%

Determination of H⁺/ATP Stoichiometry for the Plasma Membrane H⁺-ATPase from Red Beet (Beta vulgaris L.) Storage Tissue

Briskin¹,

Niesman

1991

Plant Physiol.

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“…The majority of the gastric vesicles will achieve an internal pH much less than 3.0, based on the degree of acridine orange quenching (28). It can, therefore, be assumed that the majority of the compound accumulated in the acid space is converted to the sulfenamide.…”

Section: Discussionmentioning

confidence: 99%

“…Acid Transport-Acidification of the gastric vesicles was measured by the quenching of acridine orange as described previously (28). Briefly, the vesicles at 10 g/ml were suspended in a medium containing 250 mM sucrose, 150 mM KCl, 1 M acridine orange, 5 mM Tris/HCl buffer, pH 6.8, and 1 g/ml valinomycin.…”

Section: Methodsmentioning

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

245

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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Redistribution of gastric K⁺‐NPPase in vertebrate oxyntic cells in relation to hydrochloric acid secretion: A cytochemical study

Koenig

1984

Anat. Rec.

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Gastric K+-NPPase represents a partial reaction of the (K+-H+)ATPase system, which is considered to be the proton pump in mammalian parietal cells. In the present paper, K+-NPPase activity was cytochemically studied by the method of Mayahara et al. (1980) in gastric glands of birds, amphibia, and mammals, either in the resting state induced by cimetidine or after stimulation of HCl secretion by histamine. The gastric K+-NPPase cytochemical reaction was localized only in oxyntic cells of the gastric mucosa in the three species tested. The subcellular distribution of the K+-NPPase reaction product drastically changes with the secretory state of HCl. In resting cells, the K+-NPPase staining is associated with the membranes of the endocellular tubular system while in HCl-secreting cells, it is associated with the plasma membrane of the elaborate secretory surface characteristic of this functional state. The above results demonstrate that the same enzymatic activity, which is associated with the gastric proton pump, is present in both membranous systems of the oxyntic cell secretory pole. This fact supports the proposal that the tubular system represents a membrane reserve that inserts the proton pump into the luminal plasma membrane in vertebrate oxyntic cells under the action of HCl secretagogues.

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Quantitation of hydrogen ion and potential gradients in gastric plasma membrane vesicles

Cited by 87 publications

References 26 publications

Determination of H⁺/ATP Stoichiometry for the Plasma Membrane H⁺-ATPase from Red Beet (Beta vulgaris L.) Storage Tissue

Determination of H⁺/ATP Stoichiometry for the Plasma Membrane H⁺-ATPase from Red Beet (Beta vulgaris L.) Storage Tissue

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

Redistribution of gastric K⁺‐NPPase in vertebrate oxyntic cells in relation to hydrochloric acid secretion: A cytochemical study

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Quantitation of hydrogen ion and potential gradients in gastric plasma membrane vesicles

Cited by 87 publications

References 26 publications

Determination of H+/ATP Stoichiometry for the Plasma Membrane H+-ATPase from Red Beet (Beta vulgaris L.) Storage Tissue

Determination of H+/ATP Stoichiometry for the Plasma Membrane H+-ATPase from Red Beet (Beta vulgaris L.) Storage Tissue

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

Redistribution of gastric K+‐NPPase in vertebrate oxyntic cells in relation to hydrochloric acid secretion: A cytochemical study

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Determination of H⁺/ATP Stoichiometry for the Plasma Membrane H⁺-ATPase from Red Beet (Beta vulgaris L.) Storage Tissue

Determination of H⁺/ATP Stoichiometry for the Plasma Membrane H⁺-ATPase from Red Beet (Beta vulgaris L.) Storage Tissue

Redistribution of gastric K⁺‐NPPase in vertebrate oxyntic cells in relation to hydrochloric acid secretion: A cytochemical study