Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound

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Query: EC:3.6.1.3 (ATPase)
65,361 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

A compound, SCH 28080 (2-methyl-8-(phenylmethoxy)imidazo [1,2-a]pyridine-3-acetonitrile), reversibly inhibits gastric and renal ouabain-insensitive H+,K+-ATPase, but not colonic ouabain-sensitive H+,K+-ATPase. By using the functional expression system and site-directed mutagenesis, we analyzed the putative binding sites of SCH 28080 in gastric H+,K+-ATPase alpha-subunit. It was previously reported that the binding site of SCH 28080, which is a K+-site inhibitor specific for gastric H+,K+-ATPase, was in the first extracellular loop between the first and second transmembrane segments of the alpha-subunit; Phe-126 and Asp-138 were putative binding sites. However, we found that all the mutants in the first extracellular loop including Phe-126 and Asp-138 retained H+, K+-ATPase activity and sensitivity to SCH 28080. Therefore, amino acid residues in the first extracellular loop are not directly involved in the SCH 28080 binding nor indispensable for the H+, K+-ATPase activity. Here we propose a candidate residue that is important for the binding with SCH 28080, Glu-822 in the sixth transmembrane segment. Mutations of Glu-822 to Asp and Ala (mutants termed E822D and E822A, respectively) decreased the ATPase activity to about 45% and 35% of the wild-type enzyme, respectively, while the mutations to Gln and Leu abolished the activity. Mutant E822A showed a significantly lower affinity for K+ than the wild-type enzyme, indicating that Glu-822 is involved in determining the affinity for K+. The sensitivity of mutant E822D to SCH 28080 was 8 times lower than that of the wild-type enzyme. The counterpart of Glu-822 in gastric H+,K+-ATPase is Asp in Na+,K+-ATPase and other colonic ouabain-sensitive H+,K+-ATPase, which are insensitive to SCH 28080. These results suggest that Glu-822 is one of important sites that bind with SCH 28080.
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PMID:Mutational analysis of putative SCH 28080 binding sites of the gastric H+,K+-ATPase. 921 17

We investigated the effect of a newly synthesized compound, 3-amino-5-methyl-2-(2-methyl-3-thienyl)imidazo[1,2-a]thieno[3,2-c]pyridi ne (SPI-447), on the activity of H+,K+-ATPase isolated from porcine gastric mucosa. In lyophilized gastric vesicles, SPI-447 inhibited K+-stimulated ATPase activity in a dose-dependent manner in a pH 7.4 solution (IC50=4.2 microM (1.05-16.8)). The inhibitory action of SPI-447 was enhanced at pH 6.8 (IC50=1.05 microM (0.31-3.57)) and not influenced by glutathione. In intact gastric vesicles, SPI-447 had no effect on the spontaneous diffusion of H+ across the microsomal membrane. These results indicate that SPI-447 directly inhibits gastric H+,K+-ATPase activity in a SH group-independent manner.
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PMID:Inhibition of gastric H+, K+-ATPase by 3-amino-5-methyl-2-(2-methyl-3-thienyl)imidazo[1,2-a]thieno[3,2-c]pyrid ine, SPI-447. 943 64

Models of P-type ATPase predict that membrane-embedded fragments represent about 20% of the protein and adopt an all-alpha-helical structure. While this prediction was confirmed for the Ca2-ATPase [Corbalan-Garcia, S., Teruel, J., Villalain, J. & Gomez-Fernandez, J. (1994) Biochemistry 33, 8247-8254], it is at odds with recent experimental evidence gathered on the Neurospora crassa plasma membrane H+-ATPase [Vigneron, L., Ruysschaert, J.-M. & Goormaghtigh, E. (1995) J. Biol. Chem. 270, 17685-17696] and on the gastric H+,K+-ATPase [Raussens, V., Ruysschaert, J.-M. & Goormaghtigh, E. (1997) J. Biol. Chem. 276, 262-270]. Extensive proteinase K proteolysis of open gastric tubulovesicles was performed here to generate the membrane-protected fragments of the H+,K+-ATPase. Secondary structure of the intact and of the membrane-protected segments was compared for oriented membrane films by attenuated total-reflection Fourier-transform infrared spectroscopy and by circular dichroism and for vesicles suspension by circular dichroism and Raman spectroscopy. All the spectroscopic data indicate that the protease-resistant membrane-bound residue of the H+,K+-ATPase contains significant amount of beta-sheet structure, both on films and in membrane suspensions. Polarized attenuated total-reflection infrared spectroscopy indicates that only the alpha-helical content of protease-resistant membrane-bound residue of the H+,K+-ATPase is oriented (parallel) with respect to the membrane normal. Raman spectroscopy reveals that Phe residues are preferentially removed by protease activity. Evaluation of the amount of removed Phe and Tyr residues places constraints on the model of membrane insertion of the H+,K+-ATPase.
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PMID:Secondary structure of the intact H+,K+-ATPase and of its membrane-embedded region. An attenuated total reflection infrared spectroscopy, circular dichroism and Raman spectroscopy study. 952 97

Previous experiments from our laboratory (Codina, J., Kone, B. C., Delmas-Mata, J. T., and DuBose, T. D., Jr. (1996) J. Biol. Chem. 271, 29759-29763) demonstrated that the alpha-subunit of the colonic H+, K+-ATPase (HKalpha2) requires coexpression with a beta-subunit to support H+/K+ transport in a heterologous expression system (Xenopus laevis oocytes). In these studies, HKalpha2 formed stable and functional alpha.beta complexes when coexpressed with either the rat beta1-subunit of the Na+,K+-ATPase or the beta-subunit of the gastric H+,K+-ATPase, suggesting that different beta-subunits may interact with HKalpha2. The present studies tested this hypothesis by development and application of a specific antibody against HKalpha2 peptide. Subsequently, immunoprecipitation experiments were performed to determine if HKalpha2 co-precipitates with the same beta-subunit in organs known to express HKalpha2 protein. The data demonstrate that HKalpha2 assembles with beta1-Na+,K+-ATPase in the renal medulla and in distal colon.
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PMID:The alpha-subunit of the colonic H+,K+-ATPase assembles with beta1-Na+,K+-ATPase in kidney and distal colon. 952 84

To investigate the role of Glu820, located in transmembrane domain M6 of the alpha-subunit of gastric H+,K+-ATPase, a number of mutants was prepared and expressed in Sf9 cells using a baculovirus encoding for both H+,K+-ATPase subunits. The wild-type enzyme and the E820D (Glu820-->Asp) mutant showed a similar biphasic activation by K+ on the ATPase activity (maximum at 1 mM). The mutant E820A had a markedly decreased K+ affinity (maximum at 40-100 mM). The other mutants, E820Q, E820N, E820L and E820K, showed no K+-activated ATPase activity at all, whereas all mutants formed a phosphorylated intermediate. After preincubation with K+ before phosphorylation mutant E820D showed a similar K+-sensitivity as the wild-type enzyme. The mutants E820N and E820Q had a 10-20 times lower sensitivity, whereas the other three mutants were hardly sensitive towards K+. Upon preincubation with 3-(cyanomethyl)-2-methyl-8-(phenylmethoxy) imidazo [1,2a]-pyridine (SCH28080), all mutants showed similar sensitivity for this drug as the wild-type enzyme, except mutant E820Q, which could only partly be inhibited, and mutant E820K, which was completely insensitive towards SCH28080. These experiments suggest that, with a relatively large residue at position 820, the binding of SCH28080 is obstructed. The various mutants showed a behaviour in K+-stimulated-dephosphorylation experiments similar to that for K+-activated-ATPase-activity measurements. These results indicate that K+ binding, and indirectly the transition to the E2 form, is only fully possible when a negatively charged residue is present at position 820 in the alpha-subunit.
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PMID:The negative charge of glutamic acid-820 in the gastric H+,K+-ATPase alpha-subunit is essential for K+ activation of the enzyme activity. 953 86

In the reaction cycle of P-type ATPases, an acid-stable phosphorylated intermediate is formed which is present in an intracellularly located domain of the membrane-bound enzymes. In some of these ATPases, such as Na+,K+-ATPase and gastric H+, K+-ATPase, extracellular K+ ions stimulate the rate of dephosphorylation of this phosphorylated intermediate and so stimulate the ATPase activity. The mechanism by which extracellular K+ ions stimulate the dephosphorylation process is unresolved. Here we show that three mutants of gastric H+,K+-ATPase lacking a negative charge on residue 820, located in transmembrane segment six of the alpha-subunit, have a high SCH 28080-sensitive, but K+-insensitive ATPase activity. This high activity is caused by an increased 'spontaneous' rate of dephosphorylation of the phosphorylated intermediate. A mutant with an aspartic acid instead of a glutamic acid residue in position 820 showed hardly any ATPase activity in the absence of K+, but K+ ions stimulated ATPase activity and the dephosphorylation process. These findings indicate that the negative charge normally present on residue 820 inhibits the dephosphorylation process. K+ ions do not stimulate dephosphorylation of the phosphorylated intermediate directly, but act by neutralizing the inhibitory effect of a negative charge in the membrane.
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PMID:Constitutive activation of gastric H+,K+-ATPase by a single mutation. 960 85

The sarcoplasmic reticulum Ca2+-ATPase and the gastric H+,K+-ATPase were cleaved under three different proteolysis conditions. After elimination of the protease and of the cleaved peptides, the vesicles containing the membrane-bound peptides of the ATPases were studied by Fourier transform attenuated total reflection infrared spectroscopy. In the harsher proteolysis conditions, the membrane-associated domain of the Ca2+-ATPase represented about 20% of the protein and was mainly constituted of alpha-helices. Polarized infrared spectroscopy showed that these alpha-helices were mainly oriented perpendicular to the membrane. However, only 10-20% of the H+,K+-ATPase was cleaved. The remaining, membrane-associated domain of the protein contained about 30% of alpha-helices and 30% of beta-sheet structures. The alpha-helices adopted a mainly transmembrane orientation. While the data on the Ca2+-ATPase are in general agreement with the current model of the protein, our results indicate that caution must be used in choosing this protein as a general structural model for all P-type ATPases. The protease-resistant, membrane-associated domain of the H+K+-ATPase is indeed much larger than predicted and also contained beta-sheet structures.
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PMID:Secondary structure of the membrane-bound domains of H+,K+-ATPase and Ca2+-ATPase, a comparison by FTIR after proteolysis treatment of the native membranes. 982 87

Mutagenesis of Glu820, present in the catalytic subunit of gastric H+,K+-ATPase, into an Asp hardly affects K+-stimulated ATPase and K+-stimulated dephosphorylation of the enzyme. The ATP phosphorylation rate of the E820D mutant, however, is rather low and the apparent affinity for ATP in the phosphorylation process of this mutant is 2-3 times lower than that of the wild type enzyme. The reduction in the ATP phosphorylation rate of the E820D mutant has only an effect on the ATPase activity at low temperature. These findings suggest that Glu820 might play a role in H+ stimulation of the phosphorylation process.
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PMID:Mutagenesis of glutamate 820 of the gastric H+,K+-ATPase alpha-subunit to aspartate decreases the apparent ATP affinity. 988 80

Aging is often related to electrolyte disorders. This study was designed to evaluate the renal capacity for K+ handling of aging rats submitted to both extreme situations of K+ depletion and K+ loading. Aging rats were submitted to metabolic assessment, measurement of plasma aldosterone and determination of Na+,K+-ATPase and H+,K+-ATPase activity in the inner medullary collecting duct (IMCD). During K+ depletion, aging rats showed low values of urine K+ excretion. After 2 weeks of dietary K+ restriction, the plasma K+ levels of these animals reached 2.9+/-0.1 mEq/l without acid-base disorders. During K+ loading, aging rats showed a significant increase in urinary K+ excretion, with plasma K+ remaining at normal levels (4.7+/-0.1 mEq/l). Plasma aldosterone levels were low in control aging rats but K+ intake modulated the levels of this hormone, with K+ depletion reducing it and K+ loading significantly increasing it. Na+,K+-ATPase activity increased only in the initial portion of the IMCD of aging rats after 2 weeks of K+ depletion. In the distal portion of the IMCD, Na+,K+-ATPase activity did not change. H+,K+-ATPase remained unchanged in both the proximal and distal portions of the IMCD of aging rats. During K+ loading, there was no change in Na+,K+-ATPase or H+,K+-ATPase activity in the proximal or distal portions of the IMCD of aging rats. This study suggests that the senescent kidney responded to K+ conservation and excretion under the two extremes of low and high K+ intake.
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PMID:Renal potassium handling in aging rats. 993 27

Gastric H+,K+-ATPase can be inhibited by imidazo pyridines like 2-methyl-8-[phenylmethoxy] imidazo-(1,2a) pyridine 3-acetonitrile (SCH 28080). The drug shows a high affinity for inhibition of K+-activated ATPase and for prevention of ATP phosphorylation. The inhibition by SCH 28080 can be explained by assuming that SCH 28080 binds to both the E2 and the phosphorylated intermediate (E2-P) forms of the enzyme. We observed recently that some mutants, in which glutamic acid 820 present in transmembrane domain six of the catalytic subunit had been replaced (E820Q, E820N, E820A), lost their K+-sensitivity and showed constitutive ATPase activity. This ATPase activity could be inhibited by similar SCH 28080 concentrations as the K+-activated ATPase of the wild-type enzyme. SCH 28080 also inhibited ATP phosphorylation at 21 degrees C of the mutants E820D, E820N, and E820A, although with varying efficacy and affinity. ATP-phosphorylation of mutant E820Q was not inhibited by SCH 28080; in contrast, the phosphorylation level at 21 degrees C was nearly doubled. These findings can be explained by assuming that mutation of Glu820 favors the E1 conformation in the order E820Q >E820A >E820N >wild-type = E820D. The increase in the phosphorylation level of the E820Q mutant can be explained by assuming that during the catalytic cycle the E2-P intermediate forms a complex with SCH 28080. This intermediate hydrolyzes considerably slower than E2-P and thus accumulates. The high tendency of the E820Q mutant for the E1 form is further supported by experiments showing that ATP phosphorylation of this mutant is rather insensitive towards vanadate, inorganic phosphate, and K+.
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PMID:Conformation-dependent inhibition of gastric H+,K+-ATPase by SCH 28080 demonstrated by mutagenesis of glutamic acid 820. 1005 39


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