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)

The gastric H,K-ATPase (EC 3.6.1.3) is responsible for acid secretion into the stomach and is composed of two subunits, alpha and beta. The structure of the gene encoding the mouse beta subunit and the expression of both subunits during ontogeny are reported. The gene spans approximately 12 kilobase pairs and contains 7 exons. The positions at which introns interrupt the coding regions of the mouse H,K-ATPase beta subunit and mouse Na,K-ATPase (EC 3.6.1.37) beta 2 subunit genes are identical. The alternative beta subunit isoform of the Na,K-ATPase, beta 1, has a similar but not identical gene structure. Primer extension and S1 nuclease analysis of RNA isolated from mouse stomachs aged between 2 and 25 days indicated that major transcription-initiation sites are between 22 and 25 base pairs 5' of the translation initiation site at all ages. The expression of the H,K-ATPase alpha and beta subunit genes during ontogeny (day 1-40) was found to be co-ordinated. Protein levels of both the ATPase alpha and beta subunits were very low until day 15 and then increased to adult levels by day 30. In any mucosal cell throughout ontogeny, expression of the beta subunit gene invariably coincided with the expression of the alpha subunit gene. Cells detected by anti-H,K-ATPase beta subunit antibodies in sections from 10- and 30-day-old mice all had typical morphology of parietal cells and were arranged in glandular structures. Co-ordinate expression of the two subunit genes suggest that the regulatory mechanisms will be similar and that the beta subunit may be required for localization and function of the catalytic alpha subunit.
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PMID:The mouse gastric H,K-ATPase beta subunit. Gene structure and co-ordinate expression with the alpha subunit during ontogeny. 137 Apr 59

The effects of detergents and free fatty acids on the K(+)-activated ATPase activity and on the steady-state phosphorylation level of pig gastric H,K-ATPase were studied. Unsaturated free fatty acids inhibited the K(+)-activated ATPase activity, due to inactivation of the enzyme (long-term effects) and to a decrease in the K(+)-sensitive dephosphorylation rate (short-term effects). The degree of inhibition depended on the reaction conditions: the protein concentration, the temperature and the ligands used. No effect was observed when saturated- or methylated unsaturated fatty acids were tested. Free fatty acids and the detergent C12E8 increased the steady-state ATP phosphorylation level, indicating the presence of vesicular structures in the H,K-ATPase preparations. At higher concentrations these compounds inactivated H,K-ATPase, which was measured as a decrease in phosphorylation capacity. By combining the data from the ATP phosphorylation level in the absence and presence of C12E8 (without inactivation) and the data from the K(+)-activated ATPase activity with and without ionophore the tightness of vesicular preparations and the orientation of H,K-ATPase was determined. A rather simple method for the isolation of H,K-ATPase is reported, which yields highly purified H,K-ATPase preparations with a ATP phosphorylation capacity of 3.9 nmol P per mg protein or 0.57 mol P per mol alpha beta protomer. This number suggests that each alpha-subunit H,K-ATPase can be phosphorylated at the same time.
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PMID:Effect of free fatty acids and detergents on H,K-ATPase. The steady-state ATP phosphorylation level and the orientation of the enzyme in membrane preparations. 166 35

A rat genomic library was screened using a gastric H,K-ATPase beta-subunit cDNA probe, and two clones were identified. Restriction endonuclease mapping and Southern hybridization analyses indicated that each of these clones contains the entire H,K-ATPase beta-subunit gene. The nucleotide sequence was determined for the 8.75-kb transcription unit and 2.2 kb of the 5'-flanking region. The gene consists of seven exons and shows a high degree of similarity to the Na,K-ATPase beta 1-subunit gene. Primer extension and S1 nuclease protection analyses identified a major transcription initiation site 23 bases upstream of the translation start site and several minor transcription initiation sites located further upstream. The 5'-flanking region of the gene has two potential TATA sequences, each located 25-30 bases upstream of a transcription initiation site, and a number of potential promoter and regulatory elements. In addition, the 5'-flanking region contains nucleotide sequences that may regulate transcription through the formation of unusual DNA structures. These include a sequence that may form a triple helix and an adjacent sequence with the potential to form Z-DNA.
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PMID:Rat gastric H,K-ATPase beta-subunit gene: intron/exon organization, identification of multiple transcription initiation sites, and analysis of the 5'-flanking region. 166 70

We have isolated cDNA clones encoding the bovine and rat gastric H,K-ATPase beta subunit. A bovine abomasum lambda gt11 cDNA library was screened with a monoclonal antibody raised against the rabbit H,K-ATPase beta subunit. A single positive phage clone containing an approximately 900-base pair cDNA insert was identified as reactive with the antibody. The identity of the cDNA was established by comparing the deduced amino acid sequence with sequences of cyanogen bromide fragments of the porcine H,K-ATPase beta subunit. Polymerase chain reaction and rapid amplification of cDNA ends were used to generate a cDNA fragment encoding the carboxyl-terminal portion of the rat gastric H,K-ATPase beta subunit. A rat stomach cDNA library was screened with the polymerase chain reaction product, and several full-length beta subunit cDNA clones were identified. The open reading frame predicts a protein of 294 amino acids with a molecular weight of 33,689. The rat H,K-ATPase beta subunit shows 41% amino acid sequence identity to the rat Na,K-ATPase beta 2 subunit and shares a number of structural similarities with Na,K-ATPase beta subunit isoforms. By analyzing the segregation of restriction fragment length polymorphisms among recombinant inbred strains of mice, we localized the H,K-ATPase beta subunit gene to murine chromosome 8. Northern and Western blot analysis reveals that this gene is expressed exclusively in stomach. Our results suggest that the H,K-ATPase and Na,K-ATPase beta subunits evolved from a common ancestral gene and may play similar functional roles in enzyme activity.
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PMID:Cloning of the H,K-ATPase beta subunit. Tissue-specific expression, chromosomal assignment, and relationship to Na,K-ATPase beta subunits. 197 34

A cDNA encoding the beta-subunit of the rat gastric H,K-ATPase has been identified using oligonucleotide probes based on the amino acid sequences of two peptides from the pig H,K-ATPase beta-subunit (Hall, K., Perez, G., Anderson, D., Gutierrez, C., Munson, K., Hersey, S. J., Kaplan, J. H., and Sachs, G. (1990) Biochemistry 29, 701-706). The nucleotide sequence of the 1.3-kilobase cDNA has been determined and the primary structure of the protein deduced. The protein consists of 294 amino acids and has an Mr of 33,625. The amino acid sequence of the H,K-ATPase beta-subunit is similar to those of the beta 1 (29% identity) and beta 2 (37% identity) subunits of the Na,K-ATPase. Based on the hydropathy profile it seems to have the same transmembrane organization as the Na,K-ATPase beta-subunit, with a single membrane-spanning domain near the amino terminus. Seven potential N-linked glycosylation sites are located in the putative extracellular regions of the protein. Northern blot analyses of poly(A)+ RNAs from 13 tissues demonstrate that the H,K-ATPase beta-subunit mRNA is expressed at high level in stomach and is not expressed in any of the other tissues.
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PMID:cDNA cloning of the beta-subunit of the rat gastric H,K-ATPase. 216 52

A soluble porcine H,K-ATPase preparation was obtained with the nonionic detergent, C12E8. ATP hydrolysis by the soluble H,K-ATPase was stimulated with respect to the native preparation at pH 6.1, while the K(+)-phosphatase activity was comparable to the native enzyme. The soluble enzyme demonstrated characteristic ligand-dependent effects on ATP hydrolysis, including ATP activation of K(+)-stimulated hydrolysis with a K0.5 of 28 +/- 4 microM ATP, and inhibition with an IC50 of 2.1 mM ATP. The activation and inhibition of ATP hydrolysis by K+ was also observed with a K0.5 for activation of 2.8 +/- 0.4 mM KCl at 2.0 mM ATP (pH 6.1) and inhibition with an IC50 of 135 mM KCl at 0.05 mM ATP. 2-Methyl-8-(phenylmethoxy)imidazo[1,2a]pyridine-3-acetonitrile (SCH 28080), a specific inhibitor of the native H,K-ATPase, competitively inhibited the K(+)-stimulated activity with a Ki of 0.035 microM. The soluble enzyme was stable with a t0.5 for ATPase activity of 6 h between 4 and 11 degrees C. The demonstration of these related ligand responses in the catalytic reactions of the soluble preparation indicates that it is an appropriate medium for investigation of the subunit associations of the functional H,K-ATPase. Subunit associations of the active soluble enzyme were assessed following treatment with the crosslinking reagent, glutaraldehyde. The distribution of crosslinked particles was independent of the soluble protein concentration in the crosslinking buffer within the protein range 0.3 to 2.0 mg/ml or the detergent to protein ratio varied from 1 to 15 (w/w). The crosslinked pattern was unaffected by the presence or absence of K during crosslinking or nucleotide concentration. These observations suggest that crosslinking occurs in associated subunits that do not undergo rapid associations dependent upon enzyme turnover. Phosphorylation of the soluble enzyme with 0.1 mM MgATP produced a phosphoprotein at 94 kDa. A phosphoprotein obtained after glutaraldehyde treatment exhibited identical electrophoretic mobility to the crosslinked particle identified by silver stain. Glutaraldehyde treatment of soluble protein fractions resolved on a linear 10-35% glycerol gradient revealed several smaller peptides partially resolved from the crosslinked pump particle, but no active fraction enriched in the monomeric H,K-ATPase. This data indicates that the functional porcine gastric H,K-ATPase is organized as a structural dimer.
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PMID:Glutaraldehyde crosslinking analysis of the C12E8 solubilized H,K-ATPase. 216 16

Following a recent demonstration that H,K-ATPase can active transport Na+ at a low rate (Polvani, C., Sachs, G., and Blostein, R. (1989) J. Biol. Chem. 264, 17854-17859), we have looked for and found effects of Na+ ions on the conformational state of gastric H,K-ATPase labeled with fluorescein isothiocyanate. Na+ ions reverse the K(+)-induced quench of the fluorescein fluorescence and somewhat enhance fluorescence in the absence of K+ ions. Equilibrium titrations of the cation effects show that Na+ and K+ ions are strictly competitive with apparent dissociation constants of KNa+ = 62 mM (n = 2) and KK+ = 6.6 mM (n = 2). The observations demonstrate that Na+ ions bind to and stabilize the high fluorescence E1 form of the protein while K+ ions stabilize the low fluorescence E2 form. Elevation of pH from 6.4 to 8.0 increased the apparent affinity of the Na+ ions from approximately 62 to 10.2 mM, consistent with competition between protons and Na+. The action of Na+ to stabilize the E1 form was used to measure the rate of the E2K----E1Na transition with a stopped-flow fluorimeter. The rate at pH 6.4 and 20 degrees C is 18.1 s-1. In addition the rate of the reverse conformational transition E1K----E2K has been measured at several K+ concentrations. From the hyperbolic dependence on K+ concentration a maximal rate of 211 +/- 32 s-1 and intrinsic K+ dissociation constant on E1 of 64.6 +/- 3.3 mM have been estimated. The kinetic and equilibrium data are self-consistent and thus support the proposed action of Na+ and K+ ions. Compared with Na,K-ATPase, the H,K-ATPase exhibits a lower affinity for Na+ on E1 and a much faster rate of the E2K----E1Na transition, but a similar affinity for K+ ions on E1 and rate of the transition E1K----E2K. The significance of the similarities and differences in cation specificity and rates of conformational changes of Na,K- and H,K-ATPases is discussed.
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PMID:Conformational transitions of the H,K-ATPase studied with sodium ions as surrogates for protons. 217 45

We have isolated and analyzed the genes encoding the human and rat gastric H,K-ATPase catalytic subunits. The complete sequence of the human gene, including 2.2 kb of 5'-flanking sequence, and the 5' end of the rat gene, including exons 1-4 and 2.5 kb of 5'-flanking sequence, have been determined. The human gene contains 22 exons. Its intron-exon organization is identical to that of the Na,K-ATPase gene, except that exon 6 corresponds to a fusion of exons 6 and 7 of the Na,K-ATPase gene. The transcription initiation sites of both the human and rat genes were determined by primer extension and S1 nuclease protection analyses. Comparison of the 5'-flanking regions of the human and rat genes revealed three extended regions of high sequence similarity, one of which includes a potential TATA box and other basic promoter elements beginning about 30 nucleotides upstream of the transcription start site. Other conserved sequences, including possible response elements for Ca2+ and cAMP, which are known intracellular mediators of acid secretion, are located up to 2 kb 5' to the transcription initiation site.
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PMID:Structure of the human gastric H,K-ATPase gene and comparison of the 5'-flanking sequences of the human and rat genes. 217 86

In view of the striking homology among various ion-translocating ATPases including Na,K-ATPase, Ca-ATPase, and H,K-ATPase, and the recent evidence that protons can replace cytoplasmic sodium as well as potassium in the reaction mechanism of the Na,K-ATPase (Polvani, C., and Blostein, R. (1988) J. Biol. Chem. 263, 16757-16763), we studied the role of sodium as a substitute for protons in the H,K-ATPase reaction. Using hog gastric H,K-ATPase-rich inside-out membrane vesicles we observed 22Na+ influx which was stimulated by intravesicular potassium ions (K+i) at pH 8.5 but not at pH 7.1. This sodium influx was observed in medium containing ATP and was inhibited by vanadate and SCH28080, a selective inhibitor of the gastric H,K-ATPase. At least 2-fold accumulation of sodium was observed at pH 8.5. Experiments aimed to determine the sidedness of the alkaline pH requirement for K+i-dependent sodium influx showed that K+i-activated sodium influx depends on pHout and is unaffected by changes in pHin. These results support the conclusion that sodium ions substitute for protons in the H,K-ATPase reaction mechanism and provide evidence for a similarity in ion selectivity and/or binding domains of the Na,K-ATPase and the gastric H,K-ATPase enzymes.
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PMID:Sodium ions as substitutes for protons in the gastric H,K-ATPase. 255 94

The transmembrane segments predicted for the Neurospora H-ATPase are laid out diagrammatically in Figure 10. Although the eight segments have arbitrarily been compressed into rectangles of the same size, they range in length from 20 residues (II) to 30 residues (IV and VI), so the corresponding helices must vary in length as well. Notable features of the model include the charged residues located just outside the plane of the membrane, with a clear excess of negative charges (5-, 1+) at the extracellular surface and a slight excess of positive charges (4+, 3-) at the cytoplasmic surface. There are also a conspicuous number of bulky residues (tryptophan, phenylalanine, and tyrosine) just inside the plane of the membrane. Within the bilayer, most of the helices are noticeably amphipathic, consistent with the expectation that at least some of them stack together to form a channel-like structure with a hydrophobic surface and a hydrophilic core. The charged residues predicted to lie within the membrane are listed in Table 2, which is a summary of data from eight of the P-type ATPases; the S. cerevisiae and S. pombe enzymes have not been included because they are nearly identical in this respect to the Neurospora enzyme. Interestingly, all of the ATPases have more membrane-embedded negative charges (5 to 8) than positive ones (0 to 4), a pattern that may be connected with their role as cation transporters. Certainly, other unrelated transport proteins have a rather different pattern of positive and negative charges: for example, the mammalian glucose transporter (1+, 2-), Na-glucose transporter (3+, 3-), and the E. coli lac permease (11+, 7-). The actual positioning of the negative charges in the P-type ATPases does not make it easy to single out the functionally important ones, however. The glutamyl residue in segment I is present in the fungal, plant, and Leishmania H-ATPases but not in the gastric H,K-ATPase. The same is true for the aspartate in segment II, except that it also appears in the muscle and brain Ca-ATPases. A glutamate is found at one end of segment III in the E. coli and fungal enzymes and at the other end in Arabidopsis; in segment IV, another glutamate appears in a well-conserved region in the Leishmania and mammalian enzymes but not in the bacterial, fungal, or plant ones.(ABSTRACT TRUNCATED AT 400 WORDS)
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PMID:Transmembrane segments of the P-type cation-transporting ATPases. A comparative study. 256 19


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