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)

Yeast cell surface growth is accomplished by constitutive secretion and plasma membrane assembly, culminating in the fusion of vesicles with the bud membrane. Coordination of secretion and membrane assembly has been investigated by examining the biogenesis of plasma membrane ATPase (PM ATPase) in secretion-defective (sec) strains of Saccharomyces cerevisiae. PM ATPase is synthesized as a approximately 106-kD polypeptide that is not detectably modified by asparagine-linked glycosylation or proteolysis during transit to the plasma membrane. Export of the PM ATPase requires the secretory pathway. In sec1, a mutant defective in the last step of secretion, large amounts of Golgi-derived vesicles are accumulated. Biochemical characterization of this organelle has demonstrated that PM ATPase and the secretory enzyme, acid phosphatase, are transported in a single vesicle species.
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PMID:Secretory vesicles externalize the major plasma membrane ATPase in yeast. 296 51

The recA1 mutation is a single point mutation that replaces glycine 160 of the recA polypeptide with an aspartic acid residue. The mutant recA1 protein has a greatly reduced single-stranded DNA-dependent ATPase activity at pH 7.5 compared to the wild-type protein. Interestingly, the recA1 protein does exhibit a vigorous ATPase activity at pH 6.2. To explore the molecular basis of this pH effect, we used site-directed mutagenesis to replace aspartic acid 160 of the recA1 polypeptide with an isosteric, but nonionizing, asparagine residue. The new [Asn160]recA protein catalyzes ATP hydrolysis at pH 7.5 with the same turnover number as the wild-type protein. This result suggests that the activation of the recA1 protein ATPase activity that occurs at pH 6.2 may be due, in part, to neutralization of the negatively charged aspartic acid 160 side chain. Although it is an active single-stranded DNA-dependent ATPase, the [Asn160]recA protein is unable to complement a recA deletion in vivo and is unable to carry out the three-strand exchange reaction in vitro. Further examination of ATP hydrolysis (under strand exchange conditions) revealed that the ATPase activity of the [Asn160]recA protein is strongly suppressed in the presence of Escherichia coli single-stranded DNA-binding protein (a component of the strand exchange assay), whereas the ATPase activity of the wild-type recA protein is stimulated by the E. coli protein. To account for these results, we speculate that ATP may induce specific conformational changes in the wild-type recA protein that are essential to the DNA pairing process and that these conformational changes may not occur with the [Asn160]recA protein.
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PMID:Construction of a recombinase-deficient mutant recA protein that retains single-stranded DNA-dependent ATPase activity. 296 15

cDNA complementary to mRNA coding for the beta subunit of dog renal (Na+ + K+)-ATPase has been cloned into lambda gt11 and the nucleotide sequence of the DNA has been determined. The amino acid sequence of the beta subunit polypeptide has also been deduced from the DNA. The mature form of the dog kidney beta subunit contains 302 amino acids with three potential asparagine-linked attachment sites for carbohydrate. The initiation methionine is removed during processing of the polypeptide to its mature form. Although the beta subunit is an integral membrane protein there is no signal sequence for the polypeptide, and hydropathy analysis predicts that the beta subunit polypeptide spans the cell membrane only once. Secondary structure predictions and a model for the structure of the beta subunit are proposed. DNA sequencing of the 5' non-coding region of the mRNA revealed a 200 bp inverted repeat from the coding region. Blot hybridization of a fragment of the beta subunit cDNA identified a single mRNA species of 2.7 kb in dog kidney and several rat tissues. RNA from rat liver was deficient in mRNA that hybridized to the dog kidney beta subunit cDNA, although mRNA that hybridized to an alpha subunit cDNA was detected. RNA from a human hepatoma cell line, HepG2, however, contained comparable levels of mRNA for both the alpha and the beta subunits.
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PMID:Molecular cloning and sequence analysis of the (Na+ + K+)-ATPase beta subunit from dog kidney. 303 Apr 34

Many types of human cells cultured in vitro are generally semipermissive for simian virus 40 (SV40) replication. Consequently, subpopulations of stably transformed human cells often carry free viral DNA, which is presumed to arise via spontaneous excision from an integrated DNA template. Stably transformed human cell lines that do not have detectable free DNA are therefore likely to harbor harbor mutant viral genomes incapable of excision and replication, or these cells may synthesize variant cellular proteins necessary for viral replication. We examined four such cell lines and conclude that for the three lines SV80, GM638, and GM639, the cells did indeed harbor spontaneous T-antigen mutants. For the SV80 line, marker rescue (determined by a plaque assay) and DNA sequence analysis of cloned DNA showed that a single point mutation converting serine 147 to asparagine was the cause of the mutation. Similarly, a point mutation converting leucine 457 to methionine for the GM638 mutant T allele was found. Moreover, the SV80 line maintained its permissivity for SV40 DNA replication but did not complement the SV40 tsA209 mutant at its nonpermissive temperature. The cloned SV80 T-antigen allele, though replication incompetent, maintained its ability to transform rodent cells at wild-type efficiencies. A compilation of spontaneously occurring SV40 mutations which cannot replicate but can transform shows that these mutations tend to cluster in two regions of the T-antigen gene, one ascribed to the site-specific DNA-binding ability of the protein, and the other to the ATPase activity which is linked to its helicase activity.
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PMID:Simian virus 40-transformed human cells that express large T antigens defective for viral DNA replication. 303 74

The transport of glutamate, apparently a primary energy source for Coxiella burnetii, has been examined. C. burnetii is shown to possess a pH-dependent active transport system for L-glutamate with an apparent Kt of 61.1 microM and Vmax of 8.33 pmol/s per mg at pH 3.5. Both L-glutamine and L-asparagine competitively inhibited transport of glutamate, but D-glutamate, L-aspartate, L-glutamate-gamma-methyl ester, methionine sulfoximine, or alpha-ketoglutarate did not compete. This transport system is both temperature and energy dependent. Uptake of glutamate is highly sensitive to uncouplers of oxidative phosphorylation such as 2,4-dinitrophenol and carbonyl cyanide-m-chlorophenyl hydrazone that decrease the proton motive force across the cytoplasmic membrane. ATPase inhibitors such as dicyclohexylcarbodiimide or metabolic poisons such as KCN, NaF, or arsenite were much less effective as inhibitors of glutamate transport. Uptake of glutamate did not appear to be coupled to Na+ symport as in Escherichia coli since no monovalent cation requirement could be demonstrated. Instead, the Vmax of glutamate transport showed good correlation with the transmembrane pH gradient (delta pH). From these results, we propose that L-glutamate transport by C. burnetii is energized via a proton motive force.
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PMID:pH dependence of the Coxiella burnetii glutamate transport system. 613 12

The eight subunits of the H+-ATPase of Escherichia coli are coded by the genes of the unc operon, which maps between bglB and asnA. A collection of unc mutations were transferred via P1 transduction into a strain in which lambda cI857 S7 was inserted into bglB. The lambda phage was induced, and asnA+ transducing phage that carried unc were selected. Transducing phage carrying mutations in the uncA, B, D, E, and F genes were used for complementation analysis with a collection of unc mutants, including mutants which had been reported previously but not genetically characterized. Some mutations gave a simple complementation pattern, indicating a single defective gene, whereas other mutations gave more complex patterns. Two mutants (uncE105 and uncE107) altered in the proteolipid (omega) subunit of F0 were not complemented by any of the lambda unc phage, even though both mutants had a fully functional F1 ATPase and therefore normal A and D genes. Hence, only limited conclusions can be drawn from genetic complementation alone, since it cannot distinguish normal from abnormal genes in certain classes of unc mutants. The lambda unc phage proved to be essential in characterizing several mutants defective in F0-mediated H+ translocation. The unc gene products were overproduced by heat induction of the lysogenized lambda unc phage to determine whether all the F0 subunits were in the membrane. Two mutants that gave a simple complementation pattern, indicative of one defective gene, did not assemble a three-subunit F0. The uncB108 mutant was shown to lack the chi subunit of F0 but to retain psi and omega. Trace amounts of an altered omega subunit and normal amounts of chi and psi were found in the uncE106 mutant. A substitution of aspartate for glycine at residue 58 of the protein was determined by DNA sequence analysis of the uncE gene cloned from the lambda uncE106 phage DNA. One of the omega-defective, noncomplementing mutants (uncE107) was shown to retain all three F0 subunits. The uncE gene from this mutant was also sequenced to confirm an asparagine-for-aspartate substitution at position 61 (the dicyclohexylcarbodiimide-binding site) of the omega subunit.
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PMID:Use of lambda unc transducing bacteriophages in genetic and biochemical characterization of H+-ATPase mutants of Escherichia coli. 622 7

It is possible to select transmembrane potential (delta psi)-altered mutants in Streptococcus pneumoniae on the basis of their resistance to the antifolate methotrexate. Comparison of such a mutant strain ( amiA9 ) with its parent was used to evaluate the role of delta psi in the uptake of certain amino acids. The delta psi-dependent uptake of isoleucine, leucine, valine, and asparagine showed a reduced maximum velocity of uptake, and decrease in the transport constant of the energy-dependent, delta psi-independent uptake of lysine, methionine, and glutamine was observed. No reduction of the intracellular pool of ATP or of lactate excretion could be detected in the mutant strain. Moreover, studies on membrane preparations suggest that the phenotype expressed by the amiA mutation is not a consequence of alteration of its ATPase activity or susceptibility to N,N'-dicyclohexylcarbodiimide. Therefore, it is unlikely that the amiA mutation affects the H+ F1F0 ATPase which is involved in the establishment of the proton motive force in anaerobic bacteria. We propose that another function contributes to delta psi in S. pneumoniae. The amiA gene may be the structural gene of that function.
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PMID:Characterization of a Streptococcus pneumoniae mutant with altered electric transmembrane potential. 623 66

Mutations in the H+-translocating ATPase complex (F1F0) of Escherichia coli have been described in which aspartyl-61 of the omega subunit ( uncE protein) is substituted by either glycine ( uncE105 ) or asparagine ( uncE107 ). Either substitution blocks the H+-translocation activity of the F0 sector of the complex. Here we report a difference in the effects of the two substitutions on the coupled ATPase activity of F1 bound to F0. Wild-type F1 was bound to the F0 of either mutant with affinities comparable to wild-type. The ATPase activity of F1 bound to uncE107 F0 was inhibited by 50%, whereas that bound to uncE105 F0 was not inhibited. Complementation studies with a pBR322-derived plasmid that carried the E gene of the unc operon only indicated that a single mutation in the host strain was responsible for the respective phenotypes. In mutants complemented by the uncE + plasmid, restoration of wild-type biochemical properties was only partial and may be attributed to a mixing of wild-type and mutant omega subunits in a hybrid F0 complex. The activity of membrane-bound F1 was less inhibited in the uncE +/ uncE107 hybrid. Paradoxically, complementation of uncE105 by the uncE + plasmid resulted in substantial inhibition of the activity of membrane-bound F1. The results indicate that a glycine-versus-asparagine substitution for aspartyl-61 must lead to altered conformations of omega and that these differences in conformation are important in the coupling between the F0 and F1 sectors of the complex.
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PMID:Mutations altering aspartyl-61 of the omega subunit (uncE protein) of Escherichia coli H+ -ATPase differ in effect on coupled ATP hydrolysis. 632 26

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 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


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