Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts:   Target Concepts:
Query: EC:3.6.1.3 (ATPase)
 65,361 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Experiments from other laboratories conducted with Leishmania donovani promastigote cells had earlier indicated that the plasma membrane Mg2+-ATPase of the parasite is an extrusion pump for H+. Taking advantage of the pellicular microtubular structure of the plasma membrane of the organism, we report procedures for obtaining sealed ghost and sealed everted vesicle of defined polarity. Rapid influx of H+ into everted vesicles was found to be dependent on the simultaneous presence of ATP (1 mm) and Mg2+ (1 mm). Excellent correspondence between rate of H+ entry and the enzyme activity clearly demonstrated the Mg2+-ATPase to be a true H+ pump. H+ entry into everted vesicle was strongly inhibited by SCH28080 (IC50 = approximately 40 microm) and by omeprazole (IC50 = approximately 50 microm), both of which are characteristic inhibitors of mammalian gastric H+,K+-ATPase. H+ influx was completely insensitive to ouabain (250 microm), the typical inhibitor of Na+,K+-ATPase. Mg2+-ATPase activity could be partially stimulated with K+ (20 mm) that was inhibitable (>85%) with SCH28080 (50 microm). ATP-dependent rapid efflux of 86Rb+ from preloaded vesicles was completely inhibited by preincubation with omeprazole (150 microm) and by 5,5'-dithiobis-(2-nitrobenzoic acid) (1 mm), an inhibitor of the enzyme. Assuming Rb+ to be a true surrogate for K+, an ATP-dependent, electroneutral stoichiometric exchange of H+ and K+(1:1) was established. Rapid and 10-fold active accumulation of [U-(14)C]2-deoxyglucose in sealed ghosts could be observed when an artificial pH gradient (interior alkaline) was imposed. Rapid efflux of [U-(14)C]d-glucose from preloaded everted vesicles could also be initiated by activating the enzyme, with ATP. Taken together, the plasma membrane Mg2+-ATPase has been identified as an electroneutral H+/K+ antiporter with some properties reminiscent of the gastric H+,K+-ATPase. This enzyme is possibly involved in active accumulation of glucose via a H+-glucose symport system and in K+ accumulation.
 ...
PMID:Leishmania plasma membrane Mg2+-ATPase is a H+/K+-antiporter involved in glucose symport. Studies with sealed ghosts and vesicles of opposite polarity. 1108 46

Blockade of the gastric acid pump, hydrogen-potassium adenosine triphosphatase (H+,K+-ATPase), by proton pump inhibitors (PPIs) is one of the most effective treatments for gastro-oesophageal reflux disease (GORD). In ideal terms, however, the inhibition of acid secretion should occur rapidly, on the first dose, and remain virtually complete in a dose-dependent manner. Several aspects of PPI biochemistry prevent the achievement of this ideal. PPIs target the final step of acid secretion and, due to the covalent nature of their inhibition of H+,K+-ATPase, cause suppression of acid secretion long after the drug has been eliminated. Their disadvantages stem from their mechanism of action: they require accumulation and activation in active parietal cells and have short plasma half-lives, hence a relatively slow onset of action. An extension of PPI plasma half-lives is an obvious goal, possibly via exploitation of probable differences in the metabolism of the two enantiomers (structural mirror images) present in current PPI formulations: e.g., clinical data on the S-enantiomer of omeprazole (esomeprazole) suggest some improvement in acid control. An alternative is to generate a pro-drug of a PPI; plasma levels of the PPI would thus depend on release of the active metabolite from the pro-drug, again extending drug half-life. Another area of active investigation is the development of acid-pump antagonists to inhibit acid secretion at its final step.
 ...
PMID:Improving on PPI-based therapy of GORD. 1143 May 7

The aim of this work was to develop a method for renal H+,K+-ATPase measurement based on the previously used Na+,K+-ATPase assay (Beltowski et al.: J Physiol Pharmacol.; 1998, 49: 625-37). ATPase activity was assessed by measuring the amount of inorganic phosphate liberated from ATP by isolated microsomal fraction. Both ouabain-sensitive and ouabain-resistant K+-stimulated and Na+-independent ATPase activity was detected in the renal cortex and medulla. These activities were blocked by 0.2 mM imidazolpyridine derivative, Sch 28080. The method for ouabain-sensitive H+,K+-ATPase assay is characterized by good reproducibility, linearity and recovery. In contrast, the assay for ouabain-resistant H+,K+-ATPase was unsatisfactory, probably due to low activity of this enzyme. Ouabain-sensitive H+,K+-ATPase was stimulated by K+ with Km of 0.26 +/- 0.04 mM and 0.69 +/- 0.11 mM in cortex and medulla, respectively, and was inhibited by ouabain (Ki of 2.9 +/- 0.3 microM in the renal cortex and 1.9 +/- 0.4 microM in the renal medulla) and by Sch 28080 (Ki of 1.8 +/- 0.5 microM and 2.5 +/- 0.9 microM in cortex and medulla, respectively). We found that ouabain-sensitive H+,K+-ATPase accounted for about 12% of total ouabain-sensitive activity in the Na+,K+-ATPase assay. Therefore, we suggest to use Sch 28080 during Na+,K+-ATPase measurement to block H+,K+-ATPase and improve the assay specificity. Leptin administered intraperitoneally (1 mg/kg) decreased renal medullary Na+,K+-ATPase activity by 32.1% at 1 h after injection but had no effect on H+,K+-ATPase activity suggesting that the two renal ouabain-sensitive ATPases are separately regulated.
 ...
PMID:Spectrophotometric method for the determination of renal ouabain-sensitive H+,K+-ATPase activity. 1236 94

Ouabain is a glycoside that binds to and inhibits the action of Na+,K+-ATPase. Little is known, however, about the specific requirements of the protein surface for glycoside binding. Using chimeras of gastric H+,K+-ATPase and Na+,K+-ATPase, we demonstrated previously that the combined presence of transmembrane hairpins M3-M4 and M5-M6 of Na+,K+-ATPase in a backbone of H+,K+-ATPase (HN34/56) is both required and sufficient for high affinity ouabain binding. Since replacement of transmembrane hairpin M3-M4 by the N terminus up to transmembrane segment 3 (HNN3/56) resulted in a low affinity ouabain binding, hairpin M5-M6 seems to be essential for ouabain binding. To assess which residues of M5-M6 are required for ouabain action, we divided this transmembrane hairpin in seven parts and individually replaced these parts by the corresponding sequences of H+,K+-ATPase in chimera HN34/56. Three of these chimeras failed to bind ouabain following expression in Xenopus laevis oocytes. Altogether, these three chimeras contained 7 amino acids that were specific for Na+,K+-ATPase. Individual replacement of these 7 amino acids by the corresponding amino acids in H+,K+-ATPase revealed a dramatic loss of ouabain binding for F783Y, T797C, and D804E. As a proof of principle, the Na+,K+-ATPase equivalents of these 3 amino acids were introduced in different combinations in chimera HN34. The presence of all 3 amino acids appeared to be required for ouabain action. Docking of ouabain onto a three-dimensional-model of Na+,K+-ATPase suggests that Asp804, in contrast to Phe783 and Thr797, does not actually form part of the ouabain-binding pocket. Most likely, the presence of this amino acid is required for adopting of the proper conformation for ouabain binding.
 ...
PMID:Phe783, Thr797, and Asp804 in transmembrane hairpin M5-M6 of Na+,K+-ATPase play a key role in ouabain binding. 1297 17

Erythrocytes are useful in evaluating K+ transport pathways involved in internal K+ balance. Several forms of H+,K+-ATPase have been described in nephron segments active in K+ transport. Furthermore, the activity of a ouabain-insensitive isoform of H+,K+-ATPase expressed in collecting duct cells may be modulated by acid-base status. Various assays were performed to determine if a ouabain-insensitive K+-ATPase is present in rat erythrocytes and, if so, whether it plays a role in internal K+ balance. Kinetic studies demonstrated that maximal stimulation of enzyme activity was achieved with 2.5 mM K+ at pH 7.4. Subsequent experiments were performed on erythrocyte membranes collected from animals submitted to varying degrees of K+ homeostasis: control rats, K+-depleted rats, K+-loaded rats, and rats rendered hyperkalemic due to acute renal failure. As observed in the collecting duct cell studies, there was a significant decrease in the activity of ouabain-insensitive K+-ATPase in the erythrocytes of both K+-loaded and metabolically alkalotic K+-depleted rats. However, this enzyme activity in erythrocyte membranes of rats with metabolic acidosis-related hyperkalemia was similar to that of control animals. This finding may be interpreted as resulting from two potentially modulating factors: the stimulating effect that metabolic acidosis has on K+-ATPase and the counteracting effect that hyperkalemia and uremia have on metabolic acidosis. In summary, we present evidence of a ouabain-insensitive K+-ATPase in erythrocytes, whose activity is modulated by acid-base status and K+ levels.
 ...
PMID:Erythrocytes may contain a ouabain-insensitive K+-ATPase which plays a role in internal K+ balance. 1466 63

Comparisons of the primary structures of the Na,K-ATPase alpha-isoforms reveal the existence of regions of structural divergence, suggesting that they are involved in unique functions. One of these regions is the isoform-specific region (ISR), located near the ATP binding site in the major cytoplasmic loop. To evaluate its importance, we constructed mutants of the rodent wild-type alpha1 and alpha3 isoforms in which the ISR was replaced with irrelevant sequences, i.e., the analogous region from the rat gastric H,K-ATPase catalytic subunit or a region from the human c-myc oncogene. Opossum kidney (OK) cells were transfected with wild-type rat alpha1, alpha3, or their corresponding chimeras and selected in ouabain. Introduction of either mutant produced ouabain-resistant colonies, consistent with functional expression of the chimeric protein and indicating that the ISR is not essential for overall Na,K-ATPase function. The introduced chimeras were then characterized enzymatically by measuring the relative rate of K(+) and Li(+) deocclusions. Results showed that exchanges of both alpha1 and alpha3 ISRs significantly modified the sensitivity for the enzyme to either K(+) or Li(+). Subsequent treatment of the cells with phorbol esters revealed an altered Na,K-ATPase transport in response to protein kinase C activation for the alpha1 chimeras. No changes were observed for the alpha3 isoform, suggesting that it is not sensitive to PKC regulation. These results demonstrated that the ISR plays an important role in ion deocclusion and in the response to PKC (only for the alpha1 isoform).
 ...
PMID:The isoform-specific region of the Na,K-ATPase catalytic subunit: role in enzyme kinetics and regulation by protein kinase C. 1561 11

Na+,K+-ATPase (porcine alpha/his10-beta) has been expressed in Pichia Pastoris, solubilized in n-dodecyl-beta-maltoside and purified to 70-80% purity by nickel-nitrilotriacetic acid chromatography combined with size exclusion chromatography. The recombinant protein is inactive if the purification is done without added phospholipids. The neutral phospholipid, dioleoylphosphatidylcholine, preserves Na+,K+-ATPase activity of protein prepared in a Na+-containing medium, but activity is lost in a K+-containing medium. By contrast, the acid phospholipid, dioleoylphosphatidylserine, preserves activity in either Na+- or K+-containing media. In optimal conditions activity is preserved for about 2 weeks at 0 degrees C. Both recombinant Na+,K+-ATPase and native pig kidney Na+,K+-ATPase, dissolved in n-dodecyl-beta-maltoside, appear to be mainly stable monomers (alpha/beta) as judged by size exclusion chromatography and sedimentation velocity. Na+,K+-ATPase activities at 37 degrees C of the size exclusion chromatography-purified recombinant and renal Na+,K+-ATPase are comparable but are lower than that of membrane-bound renal Na+,K+-ATPase. The beta subunit is expressed in Pichia Pastoris as two lightly glycosylated polypeptides and is quantitatively deglycosylated by endoglycosidase-H at 0 degrees C, to a single polypeptide. Deglycosylation inactivates Na+,K+-ATPase prepared with dioleoylphosphatidylcholine, whereas dioleoylphosphatidylserine protects after deglycosylation, and Na+,K+-ATPase activity is preserved. This work demonstrates an essential role of phospholipid interactions with Na+,K+-ATPase, including a direct interaction of dioleoylphosphatidylserine, and possibly another interaction of either the neutral or acid phospholipid. Additional lipid effects are likely. A role for the beta subunit in stabilizing conformations of Na+,K+-ATPase (or H+,K+-ATPase) with occluded K+ ions can also be inferred. Purified recombinant Na+,K+-ATPase could become an important experimental tool for various purposes, including, hopefully, structural work.
 ...
PMID:Purification of Na+,K+-ATPase expressed in Pichia pastoris reveals an essential role of phospholipid-protein interactions. 1570 60

Human autoimmune gastritis (AIG) is a chronic inflammatory disorder of the gastric corpus. We have defined the antigen repertoire and the functional properties of in vivo activated CD4+ T cells derived from the gastric mucosa of patients with AIG. A remarkable proportion of the CD4+ T cell clones proliferated in response to H+,K+-ATPase. Six epitopes identified in the alpha chain, and 5 in the beta chain, of gastric K+,K+-ATPase were recognized by autoreactive gastric T cell clones. The majority of the autoreactive T cell clones secreted IFN-gamma and showed a T helper 1 (Th1) profile. All clones produced TNF-alpha,provided help for B cell immunoglobulin production, expressed perforin-mediated cytotoxicity, and most induced Fas-Fas ligand-mediated apoptosis. Data suggest that activation of gastric H+,K+-ATPase-specific Th1 T cells is crucial in the pathogenesis of human gastric autoimmunity and atrophy.
 ...
PMID:Molecular specificity and functional properties of autoreactive T-cell response in human gastric autoimmunity. 1576 92

An acidic microenvironment formed by vacuolar ATPase (V-ATPase) expressed in plasma membranes of osteoclasts is thought to be indispensable for bone resorption. This study examined the efficacy of a novel V-ATPase inhibitor, FR202126, in reducing alveolar bone loss caused by experimental periodontitis in rats. FR202126 inhibited H+ transport in plasma membrane vesicles of murine osteoclasts, whereas FR202126 exerted no effect on H+ transport of mitochondrial ATPase or gastric H+,K+-ATPase, indicating that FR202126 is a specific inhibitor of V-ATPase. As expected from the mechanism, FR202126 remarkably inhibited in vitro bone resorption whatever bone resorptive factors were added. Moreover, FR202126 was also able to exert an inhibitory effect on in vivo bone resorption. Experimental periodontitis was induced by ligature wire tied around the contact between the first and second maxillary molars. Insertion of ligature wire for 7 days induced alveolar bone destruction by activating osteoclasts. Oral administration of FR202126 (u.i.d.) significantly prevented alveolar bone loss in experimental periodontitis which may offer a new approach to treatment of periodontal disease.
 ...
PMID:A novel inhibitor of vacuolar ATPase, FR202126, prevents alveolar bone destruction in experimental periodontitis in rats. 1640 38

Identification of insulators is one of the most difficult problems in functional mapping of genomes. For this reason, up to now only a few insulators have been described. In this article we suggest an approach that allows direct isolation of insulators by a simple positive-negative selection based on blocking enhancer effects by insulators. The approach allows selection of fragments capable of blocking enhancers from mixtures of genomic fragments prepared from up to 1-Mb genomic regions. Using this approach, a 1-Mb human genome locus was analyzed and eight potential insulators were selected. Five of the eight sequences were positioned in intergenic regions and two were within introns. The genes of the alpha-polypeptide H+/K+ exchanging ATPase (ATP4A) and amyloid beta (A4) precursor-like protein 1 (APLP1) within the locus studied were found to be flanked by insulators on both sides. Both genes are characterized by distinct tissue-specific expression that differs from the tissue specificity of the surrounding genes. The data obtained are consistent with the conception that insulators subdivide genomic DNA into loop domains that comprise genes characterized by similar expression profiles. Using chromatin immunoprecipitation assay, we demonstrated also that at least six of the putative insulators revealed in this work could bind the CTCF transcription factor in vivo. We believe that the proposed approach could be a useful instrument for functional analysis of genomes.
 ...
PMID:Identification, genome mapping, and CTCF binding of potential insulators within the FXYD5-COX7A1 locus of human chromosome 19q13.12. 1701 50


<< Previous 1 2 3 4 5 6 7 8 Next >>