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)

Under normal conditions, the rat collecting duct displays an H, K-ATPase activity with kinetic and pharmacological properties very close to those of the gastric H,K-ATPase. However, whether the collecting duct H,K-ATPase and the gastric enzyme are identical remains controversial. Therefore, we re-evaluated the expression of the mRNAs encoding the gastric H,K-ATPase alpha subunit in the rat nephron. For this purpose, gastric H,K-ATPase mRNAs were quantitated by RT-PCR at the level of microdissected nephron segments using known amounts of gastric H,K-ATPase cRNA as external standards. Results indicate that gastric H,K-ATPase mRNAs are undetectable (<1 copy per cell) in the glomerulus and along the proximal tubule, thick ascending limb of Henle's loop and collecting duct, although a faint expression ( approximately 400 copies per micro;g total RNA) is measurable in whole-kidney preparations. Gastric H,K-ATPase mRNA is also absent along the nephron of K-depleted rats and of rats with chronic metabolic acidosis and alkalosis. Taken with other data from the literature, these results suggest that the collecting duct of normal rats might express an H,K-ATPase similar, but not identical, to the gastric isoform.
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PMID:Re-evaluation of the expression of the gastric H,K-ATPase alpha subunit along the rat nephron. 900 Apr 35

The ouabain-sensitive, K-stimulated p-nitrophenyl phosphatase (K-pNPPase) activity, an associated activity of the Na,K-ATPase, was assayed in tentacles of the sea anemone Stichodactyla helianthus to investigate the possibility that the sea anemone Na,K-ATPase activity is an associated activity of an H,K-ATPase. Activity was maximal at pH 6.5-7.0, decreasing only slightly in acidic medium but falling abruptly in alkaline medium to 60% of maximum at pH 7.4. The pH of maximum activity was not remarkably altered in high ionic strength medium (560 mM choline chloride), but ouabain-sensitive K-pNPPase activity of both rat and sea anemone was strongly inhibited. Inhibitors of the gastric H,K-ATPase, 100 microM omeprazole and 10 microM SCH 28080, did not inhibit the ouabain-sensitive K-pNPPase activity. Activity of the sea anemone enzyme was inhibited by 10 microM ammonium vanadate, an inhibitor of P-type ATPase, and not by 2.5 mM sodium azide, an inhibitor of both F-type and V-type ATPase. Because the sea anemone K-pNPPase activity was previously found to be more sensitive to ouabain than the Na,K-ATPase activity, K(+)-ouabain antagonism was investigated and found to be relatively muted, whereas K(+)-Na+ competition was stronger than in the rat kidney.
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PMID:Effect of high ionic strength and inhibitors of H,K-ATPase on the ouabain-sensitive K-p-nitrophenylphosphatase activity in the sea anemone Stichodactyla helianthus. 922 81

To determine which amino acid sequences account for transmembrane folding of G7 receptors, the membrane domain of the rabbit cholecystokinin-A (CCK-A) G-protein-coupled receptor has been investigated by in vitro transcription/translation of two types of fusion vectors containing sequences that include putative transmembrane segments. First, the seven putative transmembrane domains of the CCK-A receptor were inserted individually into pGEM vectors beginning with the cDNA encoding the first 101 (HK-M0) or 139 (HK-M1) amino acids of the alpha subunit of the gastric H, K-ATPase. These were separated by the cDNA for the inserted transmembrane domains from the cDNA encoding the last 177 amino acids of the beta subunit of the H,K-ATPase containing five N-linked glycosylation consensus sequences (Bamberg, K., and Sachs, G. (1994) J. Biol. Chem. 269, 16909-16919). Transcription/translation of these fusion vectors in rabbit reticulocyte lysate +/- dog pancreatic microsomes followed by SDS-polyacrylamide gel electrophoresis defined the presence of signal anchor sequences in HK-M0 by glycosylation and stop transfer sequences in HK-M1 by inhibition of glycosylation. Six out of the seven putative transmembrane domains had membrane insertion signals, but no membrane insertion activity was found for the H3 segment in these vectors. To test the effect of specific upstream and downstream sequences on membrane insertion, vectors were also made starting with the cDNA encoding the N terminus of the CCK-A receptor separated from the last 177 amino acids of the H,K-ATPase beta subunit by cDNA encoding CCK-A receptor sequences of different lengths. In addition to transcription/translation, endoglycosidase H treatment was used to verify glycosylation when multiple bands were found in the presence of microsomes. The four positive charges in the loop between H1 and H2 were required for the correct orientation of the first transmembrane domain. The H3 segment acted as a stop transfer sequence only when the whole N terminus and H3 were followed by the positive charges in the cytoplasmic loop between H3 and H4. The activity of H6 as a signal anchor sequence depended on preceding positive charges. These translation data using two types of fusion vectors establish a seven-transmembrane folding model using only in vitro translation for the CCK-A receptor beginning with two signal anchor sequences and then alternating stop transfer and signal anchor insertions. Positive charges between H1 and H2, H3 and H4, and H5 and H6 function as cytoplasmic anchors in the membrane folding of this receptor.
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PMID:Identification of membrane insertion sequences of the rabbit gastric cholecystokinin-A receptor by in vitro translation. 924 25

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 alpha 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 beta subunit, of which 6 are disulfide-linked. To determine which of these was accessible to extracytoplasmic attack, the enzyme was inhibited by four different substituted 2-pyridylmethylsulfinyl benzimidazoles, 5-methoxy-2-[(4-methoxy-3, 5-dimethyl-2-pyridyl)methylsulfinyl]-1H-benzimidazole (omeprazole), 2-[(4-trifluoroethoxy-3-methyl-2-pyridyl)methylsulfinyl]-1H-ben zimida zole (lansoprazole), 5-difluoromethoxy-2-[3, 4-methoxy-2-pyridyl)methylsulfinyl]-1H-benzimidazole (pantoprazole), and 2-[(4-(3-methoxypropoxy)-3-methyl)-2-pyridyl)methylsulfinyl]-1H-++ +benzi midazole (rabeprazole), 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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PMID:Sites of reaction of the gastric H,K-ATPase with extracytoplasmic thiol reagents. 927 94

A cDNA for the GATA-6 (GATA-GT1) DNA binding protein was cloned from a library of the human gastric adenocarcinoma cell line MKN45. The deduced amino acid sequence (449 residues) indicates that the primary structure of human GATA-6 is highly homologous to that of the rat protein. The potential phosphorylation site for protein kinases (A and C), and histidine and alanine clusters are conserved. Whereas the rat H+/K+-ATPase alpha and beta subunit genes have two and three GATA protein binding sites in their promoter regions, respectively, the human alpha subunit gene has only one binding site [Maeda, M., Kubo, K., Nishi, T. and Futai, M. (1996) J. Exp. Biol. 199, 513-520]. We cloned the 5'-upstream region of the human H+/K+-ATPase beta subunit gene by genome walking and found that it also has a single GATA protein binding site near the TATA box. The GATA sites of the human alpha and beta subunit genes are recognized by the zinc finger domain of human GATA-6. The conservation of the GATA protein binding sites suggests that they are important for the gene regulation of the human and rat H+/K+-ATPase.
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PMID:GATA-6 DNA binding protein expressed in human gastric adenocarcinoma MKN45 cells. 931 13

A binding and a yeast two-hybrid analysis were carried out on the gastric H,K-ATPase to determine interactive regions of the extracytoplasmic domains of the alpha and beta subunits of this P type ATPase. Wheat germ agglutinin fractionation of fluorescein 5-maleimide-labeled tryptic fragments of detergent-solubilized H, K-ATPase showed that a fragment Leu855 to Arg922 of the alpha subunit was bound to the beta subunit. The yeast two-hybrid system showed that the region containing only a part of the seventh transmembrane segment, the loop, and part of the eighth transmembrane segment was capable of giving positive interaction signals with the ectodomain of the beta subunit. The sequence in the extracytoplasmic loop close to the eighth transmembrane segment, namely Arg898 to Thr928, was identified as being the site of interaction using this method. We deduced that the sequence Arg898 to Arg922 in the alpha subunit has strong interaction with the extracytoplasmic domain of the beta subunit. Again, using yeast two-hybrid analysis, two different sequences in the beta subunit Gln64 to Asn130 and Ala156 to Arg188 were identified as association domains in the extracytoplasmic sequence of the beta subunit. These data enable identification of major associative regions of the alpha-beta subunits of the H,K-ATPase.
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PMID:Regions of association between the alpha and the beta subunit of the gastric H,K-ATPase. 955 92

We have used expression of chimeras between the structurally related Na,K- and H,K-ATPase alpha subunits to localize regions that determine Na,K-ATPase activity. Segments of the rat Na,K-ATPase alpha1 subunit were replaced by the corresponding portions of the rat gastric H,K-ATPase alpha subunit, and the constructs were transfected into ouabain-sensitive human HEK 293 cells. Using the ability to transfer ouabain resistance as a measure of sodium pump activity, we identified segments within the sodium pump that could be replaced with proton pump sequences without the loss of biological activity. These functionally interchangeable segments encompassed approximately 75% of the amino acid differences between the two transporters. Segments that could not be exchanged mapped to three discrete regions. One region spans residues 63-117 and includes the first transmembrane (TM) segment and a portion of the amino-terminal cytoplasmic domain. The second, from residue 320 to residue 413, encompasses TM 4 and a portion of the third cytoplasmic domain, while the third region (encompassing residues 735-861 and 898-953) includes several TM domains in the carboxyl-terminal portion of the ATPase. Our results suggest that functional differences between Na,K- and H,K-ATPase, including differences in ion transport specificity, are likely to reside within these noninterchangeable segments.
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PMID:Domain swapping between Na,K- and H,K-ATPase identifies regions that specify Na,K-ATPase activity. 958 65

The alpha subunit of eukaryotic P-type ATPases has ten experimentally defined transmembrane or membrane inserted segments. The fifth and sixth of these are short, not predicted by hydropathy analysis, do not insert independently into microsomal membranes, and are readily removed after tryptic digestion and therefore may be membrane inserted sequences. Acid transport by the gastric H, K-ATPase is covalently inhibited by several substituted pyridyl methylsulfinyl benzimidazoles, such as omeprazole. These act as probes of accessible extracytoplasmic thiols because they are accumulated in the acid transporting gastric vesicles and then convert to thiol reactive, cationic tetracyclic sulfenamides. Inhibition is due mainly to disulfide formation with Cys813 or Cys822 in M5/6 and perhaps with a contribution from Cys892 in the loop between transmembrane segment (TM) 7 and TM8. Identification of the specific cysteine responsible for inhibition should be able to define the turn between M5 and M6. The gastric H,K-ATPase alpha-beta heterodimer was expressed as a fusion protein in HEK 293 cells. Transient transfection resulted in most of the protein being retained in the endoplasmic reticulum with only core glycosylation and minor activity of the ATPase evident. Stable transfection resulted in plasma membrane localization of the protein and complex glycosylation. The transfected but not the control cells displayed cation-stimulated, SCH 28080-inhibited ATPase activity and SCH 28080- and omeprazole-inhibited 86Rb uptake. The two cysteines in M5/6 and Cys892 in the TM7/8 loop were mutated to the amino acids found in the Na,K-ATPase in order to determine which of the three cysteine residues were important for benzimidazole inhibition. Mutation of one, two, or all three cysteines did not alter enzyme activity, 86Rb transport, or SCH 28080 inhibition. Only removal of Cys822 blocked omeprazole inhibition of 86Rb transport. These data suggest that Cys822 is present in a region of the enzyme most easily accessed by the cationic sulfenamide formed by omeprazole, presumably the turn between M5 and M6.
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PMID:Identification of the site of inhibition by omeprazole of a alpha-beta fusion protein of the H,K-ATPase using site-directed mutagenesis. 959 13

We previously have demonstrated that the colonic P-ATPase alpha subunit cDNA encodes an H,K-ATPase when expressed in Xenopus laevis oocytes. Besides its high level of amino acid homology (75%) with the Na,K-ATPase, the colonic H,K-ATPase also shares a common pharmacological profile with Na,K-ATPase, because both are ouabain-sensitive and Sch 28080-insensitive. These features raise the possibility that an unrecognized property of the colonic H, K-ATPase would be Na+ translocation. To test this hypothesis, ion-selective microelectrodes were used to measure the intracellular Na+ activity of X. laevis oocytes expressing various combinations of P-ATPase subunits. The results show that expression in oocytes of the colonic H,K-ATPase affects intracellular Na+ homeostasis in a way similar to the expression of the Bufo marinus Na,K-ATPase; intracellular Na+ activity is lower in oocytes expressing the colonic H,K-ATPase or the B. marinus Na,K-ATPase than in oocytes expressing the gastric H,K-ATPase or a beta subunit alone. In oocytes expressing the colonic H,K-ATPase, the decrease in intracellular Na+ activity persists when diffusive Na+ influx is enhanced by functional expression of the amiloride-sensitive epithelial Na+ channel, suggesting that the decrease is related to increased active Na+ efflux. The Na+ decrease depends on the presence of K+ in the external medium and is inhibited by 2 mM ouabain, a concentration that inhibits the colonic H,K-ATPase. These data are consistent with the hypothesis that the colonic H,K-ATPase may transport Na+, acting as an (Na,H),K-ATPase. Despite its molecular and functional characterization, the physiological role of the colonic (Na,H),K-ATPase in colonic and renal ion homeostasis remains to be elucidated.
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PMID:Does the colonic H,K-ATPase also act as an Na,K-ATPase? 960 Sep 98

Tyrosine-dependent sequence motifs are implicated in sorting membrane proteins to the basolateral domain of Madin-Darby canine kidney (MDCK) cells. We find that these motifs are interpreted differentially in various polarized epithelial cell types. The H, K-ATPase beta subunit, which contains a tyrosine-based motif in its cytoplasmic tail, was expressed in MDCK and LLC-PK1 cells. This protein was restricted to the basolateral membrane in MDCK cells, but was localized to the apical membrane in LLC-PK1 cells. Similarly, HA-Y543, a construct in which a tyrosine-based motif was introduced into the cytoplasmic tail of influenza hemagglutinin, was sorted to the basolateral membrane of MDCK cells and retained at the apical membrane of LLC-PK1 cells. A chimera in which the cytoplasmic tail of the H,K-ATPase beta subunit protein was replaced with the analogous region of the Na,K-ATPase beta subunit polypeptide was localized to both surface domains of MDCK cells. Mutation of tyrosine-20 of the H,K-ATPase beta subunit cytoplasmic sequence to an alanine was sufficient to disrupt basolateral localization of this polypeptide. In contrast, these constructs all remain localized to the apical membrane in LLC-PK1 cells. The FcRII-B2 protein bears a di-leucine motif and is found at the basolateral membrane of both MDCK and LLC-PK1 cells. These results demonstrate that polarized epithelia are able to discriminate between different classes of specifically defined membrane protein sorting signals.
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PMID:Tyrosine-based membrane protein sorting signals are differentially interpreted by polarized Madin-Darby canine kidney and LLC-PK1 epithelial cells. 975 32


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