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)

When pig stomach membrane H+,K(+)-ATPase preparations were incubated with [gamma-32P]ATP and Mg2+ with vanadate, 32P was incorporated into the alpha-chain of H+,K(+)-ATPase to a steady-state level of approximately 0.7 mol of phosphotyrosine (Tyr(P))/mol of phosphoenzyme intermediates. The addition of a membrane H+,K(+)-ATPase preparation with Mg2+ accelerated the liberation of 32P from Tyr(P) residues in the alpha-chain. Mild tosylphenylalanyl chloromethyl ketone-trypsin treatment solubilized 32P-containing peptides from the alpha-chain almost completely. A reverse-phase column chromatography of the supernatant gave two peaks of 32P-peptide with similar total radioactivities. The amino acid sequence of both peaks was shown to be Gly-Lys-Ala-Glu-Asn-Tyr-Glu-Leu-Tyr-Gln--, which is consistent with the amino-terminal sequence of the alpha-chain of H+,K(+)-ATPase deduced from cDNA from pig stomach except that the initial Met was absent. The comparison of the recovery of amino acid from each Edman cycle showed that the phosphorylation of Tyr10 occurred preceding the phosphorylation of Tyr7. These data and others suggested the presence of a novel membrane-bound enzyme system to participate in reversible phosphorylation of both Tyr residues in the alpha-chain of H+,K(+)-ATPase.
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PMID:Reversible phosphorylation of both Tyr7 and Tyr10 in the alpha-chain of pig stomach H+,K(+)-ATPase by a membrane-bound kinase and a phosphatase. 779 39

Most F1F0 type ATP synthases, including that in Escherichia coli, use H+ as the coupling ion for ATP synthesis. However, the structurally related F1F0 ATP synthase in Propionigenium modestum uses Na+ instead. The binding site for Na+ residues in the F0 sector of the P. modestum enzyme. We postulated that Na+ might interact with subunit c of F0. Subunit c of P. modestum and E. coli are reasonably homologous (19% identity) but show striking variations around the H(+)-translocating, dicyclohexylcarbodiimide-reactive carboxyl (Asp61 in E. coli). Several hydrophobic residues around Asp61 were replaced with polar residues according to the P. modestum sequence in the hope that the polar replacements might provide liganding groups for Na+. One mutant from 31 different mutation combinations did generate an active enzyme that binds Li+, the combination being V60A, D61E, A62S, and I63T. Li+ binding was detected by Li+ inhibition of ATP-driven H+ transport, Li+ inhibition of F1F0-ATPase activity, and Li+ inhibition of F0-mediated H+ transport. The Li+ effects were observed with membrane vesicles prepared from a delta nhaA, delta nhaB mutant background which lacks Na+/H+ antiporters, and with purified, reconstituted preparations of F0 prepared from this background strain. Li+ inhibition was observed at pH 8.5 but not at pH 7.0. H+ thus appears to compete with Li+ for the binding site. Li+ binding was abolished by replacement of Glu61 by Asp or Ser62 by Ala. The side chains at Ala60 and Thr63 may act in a supporting structural role by providing a more flexible conformation for the Li+ binding cavity. Thr63 does not appear to provide a liganding group since H+ transport in two other mutants, with Gly or Ala in place of Thr63, was also inhibited by Li+. We suggest that a X-Glu-Ser-Y or X-Glu-Thr-Y sequence may provide a general structural motif for monovalent cation binding, and that the flexibility provided by residues X and Y will prove crucial to this structure.
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PMID:Changing the ion binding specificity of the Escherichia coli H(+)-transporting ATP synthase by directed mutagenesis of subunit c. 781 24

The roles of the hydrophobic side chains of residues Phe760, Ile761, Tyr763, Leu764, and Ile765 located at the M5S5 boundary of the Ca(2+)-ATPase of sarcoplasmic reticulum were analyzed by site-directed mutagenesis. Substitution of Tyr763 with glycine resulted in a new phenotypic variant of the Ca(2+)-ATPase that catalyzed a high rate of Ca(2+)-activated ATP hydrolysis without net accumulation of Ca2+ in the microsomal vesicles. The ATPase activity of the Tyr763-->Gly mutant displayed characteristics similar to the ATPase activity of the wild-type enzyme measured in the presence of calcium ionophore, and the mutant was able to form the ADP-insensitive phosphoenzyme intermediate. Mutants Phe760-->Gly, Ile761-->Gly, Leu764-->Gly, and Ile765-->Gly were able to accumulate Ca2+. In mutants Leu764-->Gly and Ile765-->Gly, the turnover rate was low due to inhibition of dephosphorylation of the ADP-insensitive phosphoenzyme intermediate. On the other hand, mutant Leu764-->Lys dephosphorylated rapidly. Mutants Phe760-->Gly and Leu764-->Lys displayed apparent Ca2+ affinities that were reduced two and three orders of magnitude, respectively, relative to that of the wild-type.
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PMID:Functional consequences of alterations to amino acids at the M5S5 boundary of the Ca(2+)-ATPase of sarcoplasmic reticulum. Mutation Tyr763-->Gly uncouples ATP hydrolysis from Ca2+ transport. 782 30

The universally conserved DnaK and DnaJ molecular chaperone proteins bind in a coordinate manner to protein substrates to prevent aggregation, to disaggregate proteins, or to regulate proper protein function. To further examine their synergistic mechanism of action, we constructed and characterized two DnaJ deletion proteins. One has an 11-amino-acid internal deletion that spans amino acid residues 77-87 (DnaJ delta 77-87) and the other amino acids 77-107 (DnaJ delta 77-107). The DnaJ delta 77-87 mutant protein, was normal in all respects analyzed. The DnaJ delta 77-107 mutant protein has its entire G/F (Gly/Phe) motif deleted. This motif is found in most, but not all DnaJ family members. In vivo, DnaJ delta 77-107 supported bacteriophage lambda growth, albeit at reduced levels, demonstrating that at least some protein function was retained. However, DnaJ delta 77-107 did not exhibit other wild type properties, such as proper down-regulation of the heat-shock response, and had an overall poisoning effect of cell growth. The purified DnaJ delta 77-107 protein was shown to physically interact and stimulate DnaK's ATPase activity at wild type levels, unlike the previously characterized DnaJ259 point mutant (DnaJH33Q). Moreover, both DnaJ delta 77-107 and DnaJ259 bound to substrate proteins, such as sigma 32, at similar affinities as DnaJ+. However, DnaJ delta 77-107 was found to be largely defective in activating the ATP-dependent substrate binding mode of DnaK. In vivo, the ability of the mutant DnaJ proteins to down-regulate the heat-shock response was correlated only with their in vitro ability to activate DnaK to bind sigma 32, in an ATP-dependent manner, and not with their ability to bind sigma 32. We conclude, that although the G/F motif of DnaJ does not directly participate in the stimulation of DnaK's ATPase activity, nevertheless, it is involved in an important manner in modulating DnaK's substrate binding activity.
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PMID:The conserved G/F motif of the DnaJ chaperone is necessary for the activation of the substrate binding properties of the DnaK chaperone. 783 43

Transmembrane segments 1 and 2 of the yeast plasma membrane H(+)-ATPase are believed to form a helical hairpin structure that is joined by a short extracytoplasmic loop. The hairpin head region (Ala135-Phe144) was probed using site-directed mutagenesis. Scanning alanine mutagenesis produced functional H(+)-ATPase at all positions except Leu138, Asp143, and Phe144. D140A and V142A gave strong hygromycin B resistance and low pH sensitivity suggesting a major kinetic defect in these mutant enzymes. Other amino acid substitutions, such as L138Y, were highly perturbing, while mutations S139E and D140E produced minor effects on phenotype. Small uncharged residues Gly and Ala, which were inserted between Leu138 and Ser139 to examine the importance of loop length on H(+)-ATPase function, were well tolerated, while the insertion of a polar Ser residue was highly perturbing. Other additions were not tolerated by the enzyme. These results suggest that the turn region has limited structural flexibility. The conserved Phe144 residue could be changed to Trp with a minor effect on phenotype. However, neither Tyr, Arg, nor small hydrophobic residues could substitute, suggesting that this region is closely packed and hydrophobic. ATP hydrolysis measurements showed that Vmax was significantly reduced in nearly all mutant enzymes, except D140E; whereas, Km values were nearly normal. Vanadate-sensitivity and pH profiles for ATP hydrolysis were nearly normal for all mutant enzymes except insertion mutant S138+. Mutants with extreme phenotypes (S138+, Tyr138) showed significantly altered medium acidification profiles. These results support the notion that the hairpin head region linking transmembrane segments 1 and 2 forms a tightly packed conformationally sensitive domain that is coupled to the catalytic ATP hydrolysis domain.
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PMID:Mutational analysis of the first extracellular loop region of the H(+)-ATPase from Saccharomyces cerevisiae. 792 48

The glutamic acid residue Glu771 in the fifth transmembrane segment M5 of the Ca(2+)-ATPase of rabbit fast twitch muscle sarcoplasmic reticulum was substituted with lysine, alanine, and glycine by site-directed mutagenesis. Mutant Glu771-->Lys was unable to occlude Ca2+, and Ca2+ did not inhibit phosphorylation from P(i) or activate phosphorylation from ATP of this mutant. Mutants Glu771-->Ala and Glu771-->Gly were likewise unable to occlude Ca2+, but Ca2+ in the millimolar concentration range activated phosphorylation from ATP and inhibited phosphorylation from P(i) of these mutants. The dephosphorylation of the ADP-insensitive E2P phosphoenzyme intermediate of mutants Glu771-->Ala and Glu771-->Gly was found to be blocked, whereas the dephosphorylation proceeded rapidly for mutant Glu771-->Lys. This finding suggests a role of the positive charge of the lysine in induction of dephosphorylation, supporting the hypothesis that the side chain of Glu771 participates in the countertransport of two protons per Ca(2+)-ATPase cycle.
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PMID:Mutational analysis of Glu771 of the Ca(2+)-ATPase of sarcoplasmic reticulum. Effect of positive charge on dephosphorylation. 795 9

P-type ATPases constitute a large family of cation pumps that play crucial physiological roles in many organisms, including bacteria, plants and mammals. They are postulated to play important roles in a variety of environmental adaptation systems. Recently, we cloned two distinct putative P-type ATPase genes (pacS and pacL) from a photosynthetic cyanobacterium, Synechococcus species PCC7942. In this study, one of the gene products (named PacS) was found to possess a putative metal-binding motif (Gly-Met-X-Cys-X-X-Cys) in its N-terminal portion. Thus we supposed that this ATPase may function as a metal pump. Indeed, the results of Northern blotting analysis showed that pacS-mRNA specifically increases upon addition of copper or silver to the growth medium. The results of Western blotting analysis confirmed the view that PacS accumulates in copper-treated Synechococcus cells. Thus we concluded that the expression of PacS ATPase is regulated in response to the change in concentration of external metals, namely copper and silver. Consistent with this, an insertional inactivation mutant of pacS exhibited hypersensitivity in terms of growth to these potentially toxic metals. It was also revealed that PacS was mainly located in the thylakoid membrane, in which the photosynthetic reactions take place. This P-type ATPase in the thylakoid membrane is implicated as a copper-transporting system that may be involved in copper-homeostasis crucial to the photosynthetic thylakoid function.
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PMID:A copper-transporting P-type ATPase found in the thylakoid membrane of the cyanobacterium Synechococcus species PCC7942. 798 14

The mechanism of Ca++ mobilization induced by endothelins (ETs) and the receptor subtype responsible for this effect were examined in the endothelium of rabbit aortic valve. In the endothelium loaded with fura-2, ET-1 (1-100 nM) induced large transient increase followed by small sustained increase in cytosolic Ca++ level ([Ca++]i) in a concentration-dependent manner. ET-3 induced only a small increase in [Ca++]i at higher concentrations (100-300 nM) than ET-1, whereas a selective ETB agonist, 100 nM IRL 1620 (succinyl-[Glu9, Ala11,15]ET-1 8-21), was ineffective. A selective ETA antagonist, 3 microM BQ-123, (cyclo [-Asp-Pro-Val-Leu-Trp-]) but not a selective ETB antagonist, 10 microM RES-701-1 [cyclic (Gly1-Asp9) (Gly-Asn-Trp-His-Gly-Thr-Ala-Pro-Asp- Trp-Phe-Phe-Asn-Tyr-Tyr-Trp)], inhibited the effects of ET-1 and ET-3. The sustained increase in [Ca++]i induced by ET-1 was abolished by 30 microM La , although 100 nM nicardipine was ineffective. In the absence of external Ca++ (with 0.5 mM EGTA), ET-1 induced only a transient increase in [Ca++]i, which was inhibited by an inhibitor of Ca+(+)-ATPase in endoplasmic reticulum, 1 microM thapsigargin. However, an inhibitor and an activator of Ca+(+)-induced Ca+(+)-release channel, 10 microM ryanodine and 10 mM caffeine, did not change [Ca++]i. These results suggest that, in the endothelium of rabbit aortic valve, only the ETA receptor mediates the effects of ETs to increase [Ca++]i, which is attributable to the release of Ca++ from thapsigargin-sensitive and ryanodine-insensitive Ca++ stores and also to the Ca++ influx through La (-)sensitive and dihydropyridine-insensitive Ca++ channels.
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PMID:Ca++ mobilization mediated by endothelin ETA receptor in endothelium of rabbit aortic valve. 799 47

Using the chicken sarcoplasmic/endoplasmic reticulum Ca2+ (SERCA)-ATPase as a parental molecule and replacing various portions with the corresponding portions of the chicken Na+,K(+)-ATPase alpha 1 subunit, Ca2+/thapsigargin- and Na+/ouabain-sensitive domains critical for these P-type ATPase activities were identified. In the chimera, [n/c]CC, the amino-terminal amino acids Met-1 to Asp-162 of the SERCA (isoform 1) (SERCA1) ATPase were replaced with the corresponding portion (Met-1-Asp-200) of the Na+,K(+)-ATPase alpha 1 subunit. In the chimera CC[c/n], the carboxyl-terminal amino acids (Ser-830 to COOH) of the SERCA1 ATPase were replaced with the corresponding segment (Leu-861 to COOH) of the Na+,K(+)-ATPase alpha 1 subunit, and in the chimera CNC, the middle part (Gly-354-Lys-712) of the SERCA1 ATPase was exchanged with the Na+,K(+)-ATPase alpha 1 subunit (Gly-378-Lys-724). None of the chimeric molecules exhibited any detectable ouabain-sensitive Na+,K(+)-ATPase activity, but they did exhibit thapsigargin-sensitive Ca(2+)-ATPase activity. Therefore, the segments Ile-163-Gly-354 and Lys-712-Ser-830 of the SERCA1 ATPase are sufficient for Ca2+ and thapsigargin sensitivity. The SERCA1-ATPase activity of [n/c]CC, but not of CCC, CNC, or CC[c/n], was further stimulated by addition of Na+ in the assay medium containing Ca2+. This additional stimulation of SERCA1-ATPase activity by Na+ was abolished when the amino-terminal region (Met-1-Leu-69) of [n/c]CC was deleted ([delta n/c]CC). In the absence of Na+, the SERCA1-ATPase activity of [n/c]CC was inhibited by ouabain, and, in the presence of Na+, its activity was stimulated by this drug. On the other hand, the ATPase activity of [delta n/c]CC was not affected by ouabain, although [delta n/c]CC can still bind [3H]ouabain. These results suggest that a distinct Na(+)-sensitive domain (Na+ sensor) located within the restricted amino-terminal region (Met-1-Leu-69) of the Na+,K(+)-ATPase alpha 1 subunit regulates ATPase activity. The Na+ sensor also controls ouabain action in concert with the major ouabain-binding region between Ala-70 and Asp-200 of alpha 1 subunit.
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PMID:Na(+)-, ouabain-, Ca(2+)-, and thapsigargin-sensitive ATPase activity expressed in chimeras between the calcium and the sodium pump alpha subunits. 801 22

The effects of two suppressors of the defense reactions of host plants, which had been purified from the pea pathogen Mycosphaerella pinodes, as well as the effects of peptide moieties, on the ATPase activity in pea plasma membranes were examined in vitro. One of the suppressors, Supprescin B, inhibited the ATPase activity in a non-competitive manner, but the other suppressor, Supprescin A, did not. Supprescin A was observed to reduce the inhibitory effect of Supprescin B. A tripeptide, Ser-Ser-Gly, and a hexapeptide, Ser-Ser-Gly-Asp-Glu-Thr, which were the respective peptide moieties of Supprescin A and B, inhibited the ATPase activity in a competitive manner. Supprescin B and fragments of the hexapeptide, such as Asp-Glu-Thr and Gly-Asp-Glu, inhibited not only the ATPase activity but also the acid phosphatase activity of plasma membranes in vitro. These results indicate that the acidic amino-acid residues of the "Asp-Glu" moiety seem to act as inhibitors of the phosphatase activity. Thus, the peptide moiety of Supprescin B consists of at least two functional elements.
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PMID:Inhibition of ATPase activity in pea plasma membranes by fungal suppressors from Mycosphaerella pinodes and their peptide moieties. 801 82


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