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 effect of endothelins (ET-1 and ET-3) on 86Rb+ uptake as a measure of K+ uptake was investigated in cultured rat brain capillary endothelium. ET-1 or ET-3 dose-dependently enhanced K+ uptake (EC50 = 0.60 +/- 0.15 and 21.5 +/- 4.1 nM, respectively), which was inhibited by the selective ETA receptor antagonist BQ 123 (cyclo-D-Trp-D-Asp-Pro-D-Val-Leu). Neither the selective ETB agonists IRL 1620 [N-succinyl-(Glu9,-Ala11,15)-ET-1] and sarafotoxin S6c, nor the ETB receptor antagonist IRL 1038 [(Cys11,Cys15)-ET-1] had any effect on K+ uptake. Ouabain (inhibitor of Na+,K(+)-ATPase) and bumetanide (inhibitor of Na(+)-K(+)-Cl- cotransport) reduced (up to 40% and up to 70%, respectively) the ET-1-stimulated K+ uptake. Complete inhibition was seen with both agents. Phorbol 12-myristate 13-acetate (PMA), activator of protein kinase C (PKC), stimulated Na+,K(+)-ATPase and Na(+)-K(+)-Cl- cotransport. ET-1- but not PMA-stimulated K+ uptake was inhibited by 5-(N-ethyl-N-isopropyl)amiloride (inhibitor of Na+/H+ exchange system), suggesting a linkage of Na+/H+ exchange with ET-1-stimulated Na+,K(+)-ATPase and Na(+)-K(+)-Cl- cotransport activity that is not mediated by PKC.
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PMID:Endothelin 1 stimulates Na+,K(+)-ATPase and Na(+)-K(+)-Cl- cotransport through ETA receptors and protein kinase C-dependent pathway in cerebral capillary endothelium. 756 53

Glu-beta 185 of the Escherichia coli H(+)-ATPase (ATP synthase) beta subunit was replaced by 19 different amino acid residues. The rates of multisite (steady state) catalysis of all the mutant membrane ATPases except Asp- beta 185 were less than 0.2% of the wild type one; the Asp- beta 185 enzyme exhibited 15% (purified) and 16% (membrane-bound) ATPase activity. The purified inactive Cys- beta 185 F1-ATPase recovered substantial activity after treatment with iodoacetate in the presence of MgCl2; maximal activity was obtained upon the introduction of about 3 mol of carboxymethyl residues/mol of F1. The divalent cation dependences of the S-carboxymethyl- beta 185 and Asp- beta 185 ATPase activities were altered from that of the wild type. The Asp- beta 185, Cys- beta 185, S-carboxymethyl-beta 185, and Gln- beta 185 enzymes showed about 130, 60, 20, and 50% of the wild type unisite catalysis rates, respectively. The S-carboxymethyl- beta 185 and Asp- beta 185 enzymes showed altered divalent cation sensitivities, and the S-carboxymethyl- beta 185 enzyme showed no Mg2+ inhibition. Unlike the wild type, the two mutant enzymes showed low sensitivities to azide, which stabilizes the enzyme Mg-ADP complex. These results suggest that Glu- beta 185 may form a Mg2+ binding site, and its carboxyl moiety is essential for catalytic cooperativity. Consistent with this model, the bovine glutamate residue corresponding to Glu- beta 185 is located close to the catalytic site in the higher order structure (Abrahams, J.P., Leslie, A.G.W., Lutter, R ., and Walker, J.E. (1994) Nature 370, 621-628)
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PMID:Beta subunit Glu-185 of Escherichia coli H(+)-ATPase (ATP synthase) is an essential residue for cooperative catalysis. 759 42

Magnetic isolation of endocytic vesicles from Dictyostelium discoideum was accomplished after feeding the amoebae with iron oxide particles. Proteins associated with the endocytic vesicles were resolved by SDS-PAGE and digested 'in-gel' with endoproteinase Lys-C or Asp-N to generate peptides for amino acid sequencing. This strategy allowed the identification of the major protein constituents of the vesicles: namely, the A, B, D, E and 110 kDa subunits of a vacuolar type H(+)-ATPase, actin, a Rab 7-like GTPase, a p34 protein corresponding to a new cysteine proteinase and the 25 kDa product of a recently sequenced D. discoideum open reading frame.
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PMID:Identification of major proteins associated with Dictyostelium discoideum endocytic vesicles. 759 93

Modification of aspartic acid 369 in the sheep alpha 1 Na+,K(+)-ATPase to asparagine results in a membrane-associated form of Na+,K(+)-ATPase that can bind [3H]ouabain with high affinity in the presence of Mg2+ alone (KD = 20.4 +/- 2.6 nM). Ouabain binding to the D369N mutant is not stimulated by inorganic phosphate, confirming that Asp369 is both the catalytic phosphorylation site and the only Pi interaction site which stimulates ouabain binding. Cation inhibition of Mg(2+)-stimulated ouabain binding to the D369N mutant demonstrated that three Na+ and two K+ ions inhibit [3H]ouabain binding and suggests that this inhibition must occur via a cation-sensitive conformational change which does not directly involve dephosphorylation of the enzyme. In the presence of 10 mM Mg2+, ATP stimulates ouabain binding to the wild type protein, (AC50 = 21.4 +/- 2.7 microM) but inhibits the binding to the D369N mutant (IC50 = 2.52 +/- 0.17 microM) indicating that the mutation does not destroy the high affinity site for MgATP but does change the nature of the protein conformation normally induced by a nucleotide-Na+,K(+)-ATPase interaction. Increasing the Mg2+ from 1 to 10 mM did not alter the AC50 or IC50 values for ATP and reveals that the Mg2+ interaction which stimulates ouabain binding in the absence of nucleotide involves a distinct divalent cation site not associated with the binding of the magnesium-nucleotide complex. Thus, altering the catalytic phosphorylation site of Na+,K(+)-ATPase does not affect the expression of the ouabain-sensitive protein in the membrane fraction of NIH 3T3 cells and does not disrupt the binding of Na+, K+, Mg2+, ouabain, or ATP to the enzyme. However, the D369N substitution does inhibit the formation of a nucleotide-protein complex with high affinity for ouabain.
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PMID:Amino acid replacement of Asp369 in the sheep alpha 1 isoform eliminates ATP and phosphate stimulation of [3H]ouabain binding to the Na+, K(+)-ATPase without altering the cation binding properties of the enzyme. 760 86

The sarcoplasmic reticulum ATPase segment extending from the phosphorylation site (Asp-351) to the preceding transmembrane helix M4 (which is involved in Ca2+ binding in conjunction with transmembrane helices M5, M6, and M8) retains a marked sequence homology to the corresponding segments of other cation ATPases. We made 26 point mutations in this segment and found that nonconservative mutations of residues that are homologous in various cation ATPases result in strong inhibition of catalytic and transport function. Mutations of nonhomologous residues to match the corresponding residues of other cation ATPases are not inhibitory and, in some cases, produce higher activity. The inhibitory mutations affect the phosphorylated intermediate turnover, which is associated with the vectorial translocation of bound Ca2+. The same mutations do not affect the kinetics of ATPase activation by Ca2+ following enzyme preincubation with EGTA. This suggests that activation of the phosphoryl transfer reaction by Ca2+ binding and vectorial displacement of bound Ca2+ by enzyme phosphorylation do not occur simply as the forward and reverse directions of the same process, but are linked to distinct structural features of the enzyme. The peptide segment extending from the phosphorylation site in the enzyme extramembranous headpiece through the M4 helix in the membrane-bound region sustains a prominent role in transmission of the phosphorylation signal for displacement of bound Ca2+. A critical structural role of this segment is also demonstrated by the interference of specific mutations with membrane assembly of the expressed protein.
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PMID:Mutational analysis of the peptide segment linking phosphorylation and Ca(2+)-binding domains in the sarcoplasmic reticulum Ca(2+)-ATPase. 760 96

The homodimeric SecA protein is the peripheral subunit of the translocase, and couples the hydrolysis of ATP to the translocation of precursor proteins across the bacterial cytoplasmic membrane. The high affinity ATP binding activity of SecA resides in the amino-terminal domain of SecA. This domain contains a tandem repeat of the "so-called" Walker B-motif, hXhhD (Walker, J.E., Saraste, M., Runswick, M.J., and Gay, N.J. (1982) EMBO J. 1, 945-951), that in combination with motif A is responsible for the Mg(2+)-phosphate protein interaction. Two aspartate residues at positions 207 and 215 of the Bacillus subtilis SecA, and Asp-217 in the Escherichia coli SecA, that could be Mg2+ ion ligands, were individually mutated to an asparagine. Mutant SecA proteins were unable to growth-complement an E. coli secA amber mutant strain, and the E. coli SecA mutant interfered with the translocation of precursor proteins in vivo. B. subtilis mutant SecA proteins were expressed to a high level and purified to homogeneity. The high affinity ATP and Mg(2+)-ion binding activity was reduced in the Asp-207 mutant, and completely lost in the Asp-215 mutant. Both SecA proteins were defective in lipid-stimulated ATPase activity. Proteolytic studies suggest that the two subunits of the mutated dimeric SecA proteins are present in different conformational states. These data suggest that Asp-207 and Asp-215 are involved in the binding of the Mg(2+)-ion when Mg(2+)-ATP is bound to SecA, while Asp-207 fulfills an additional catalytic role, possibly in accepting a proton during catalysis.
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PMID:Identification of the magnesium-binding domain of the high-affinity ATP-binding site of the Bacillus subtilis and Escherichia coli SecA protein. 764 57

The highly conserved motif of Saccharomyces cerevisiae H(+)-ATPase 474KGAP has been proposed to participate in the formation of the phosphorylated intermediate during the catalytic cycle (Portillo, F., and Serrano, R. (1988) EMBO J. 7, 1793-1798). In addition, Lys-474 is the FITC binding site of the yeast enzyme (Portillo, F. and Serrano, R. (1989) Eur. J. Biochem. 186, 501-507). We have performed an intragenic suppressor analysis of the K474R mutation to identify the interacting regions involved in these functions. Random in vitro mutagenesis of the K474R allele resulted in seven suppressor (second-site) mutations. One mutation (V396I), located 18 residues away from the Asp-378 residue, which is phosphorylated during catalysis, is allele-specific. This provides genetic evidence of a direct interaction between the KGAP motif and the phosphorylation domain during the catalytic cycle. Three mutations (V484I, V484I/E485K, and E485K/E486K) are located near Lys-474 and may compense the structural alteration introduced by the K474R mutation. Two substitutions at the end of the predicted transmembrane stretch 2 (A165V and V169I/D170N) and another in the predicted ATP binding domain (P536L) may act as allele-nonspecific suppressors, as they are also able to suppress a mutation at the enzyme's carboxyl terminus.
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PMID:Genetic analysis of the fluorescein isothiocyanate binding site of the yeast plasma membrane H(+)-ATPase. 772 67

Mutants of each of the four divalent cation binding sites of chicken skeletal muscle troponin C (TnC) were constructed using site-directed mutagenesis to convert Asp to Ala at the first coordinating position in each site. With a view to evaluating the importance of site-site interactions both within and between the N- and C-terminal domains, in this study the mutants are examined for their ability to associate with other components of the troponin-tropomyosin regulatory complex and to regulate thin filaments. The functional effects of each mutation in reconstitution assays are largely confined to the domain in which it occurs, where the unmutated site is unable to compensate for the defect. Thus the mutants of sites I and II bind to the regulatory complex but are impaired in ability to regulate tension and actomyosin ATPase activity, whereas the mutants of sites III and IV regulate activity but are unable to remain bound to thin filaments unless Ca2+ is present. When all four sites are intact, free Mg2+ causes a 50-60-fold increase in TnC's affinity for the other components of the regulatory complex, allowing it to attach firmly to thin filaments. Calcium can replace Mg2+ at a concentration ratio of 1:5000, and at this ratio the Ca2.TnC complex is more tightly bound to the filaments than the Mg2.TnC form. In the C-terminal mutants, higher concentrations of Ca2+ (above tension threshold) are required to effect this transformation than in the recombinant wild-type protein, suggesting that the mutants reveal an attachment mediated by Ca2+ in the N-domain sites.
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PMID:Concerted action of the high affinity calcium binding sites in skeletal muscle troponin C. 773 Mar 55

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

By combining the tools of site-directed mutagenesis and [3H]ouabain binding, the functional role of glutamic acid 327 in the fourth transmembrane domain of the sheep alpha 1 isoform of Na+,K(+)-ATPase was examined with respect to its interactions with ouabain, Na+,K+,Mg2+, and inorganic phosphate. Using site-directed mutagenesis, this glutamic acid was substituted with alanine, aspartic acid, glutamine, and leucine. The mutant proteins were constructed in a sheep alpha 1 protein background such that [3H]ouabain binding could be utilized as a highly specific probe of the exogenous protein expressed in NIH 3T3 cells. Na+ competition of [3H]ouabain binding to the mutant forms of Na+,K(+)-ATPase revealed only slight alterations in their affinities for Na+ and in their abilities to undergo Na(+)-induced conformational changes which inhibit ouabain binding. In contrast, K+ competition of [3H]ouabain binding to all four mutant forms of Na+,K(+)-ATPase displayed severely altered interactions between these proteins and K+. Interestingly, [3H]ouabain binding to the mutant E327Q was not inhibited by the presence of K+. This mutant was previously reported to be functionally able to support cation transport with a 5-fold reduced K0.5 for K(+)-dependent ATPase activity (Jewell-Motz, E. A., and Lingrel, J.B. (1993) Biochemistry 32, 13523-13530; Vilsen, B. (1993) Biochemistry 32, 13340-13349). Thus, it appears that this glutamic acid in the fourth transmembrane domain may be important for stabilizing a K(+)-induced conformation within the catalytic cycle of Na+,K(+)-ATPase that is not rate-limiting in the overall ATPase cycle but that displays a greatly reduced affinity for ouabain.
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PMID:Glutamic acid 327 in the sheep alpha 1 isoform of Na+,K(+)-ATPase stabilizes a K(+)-induced conformational change. 785 79


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