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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 amino acid sequence of the proteolipid subunit of the ATP synthase was analyzed in six mutant strains from Escherichia coli K12, selected for their increased resistance towards the inhibitor N,N'-dicyclohexylcarbodiimide. All six inhibitor-resistant mutants were found to be altered at the same position of the proteolipid, namely at the isoleucine at residue 28. Two substitutions could be identified. In type I this residue was substituted by a valine resulting in a moderate decrease in sensitivity to dicyclohexylcarbodiimide. Type II contained a threonine residue at this position. Here a strong resistance was observed. These two amino acid substitutions did not influence functional properties of the ATPase complex. ATPase as well as ATP-dependent proton-translocating activities of mutant membranes were indistinguishable from the wild type. At elevated concentrations, dicyclohexylcarbodiimide still bound specifically to the aspartic acid at residue 61 of the mutant proteolipid as in the wild type, and thereby inhibited the activity of the ATPase complex. It is suggested that the residue 28 substituted in the resistant mutants interacts with dicyclohexylcarbodiimide during the reactions leading to the covalent attachment of the inhibitor to the aspartic acid at residue 61. This could indicate that these two residues are in close vicinity and would thus provide a first hint on the functional conformation of the proteolipid. Its polypeptide chain would have to fold back to bring together these two residues separated by a segment of 32 residues.
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PMID:Identification of amino-acid substitutions in the proteolipid subunit of the ATP synthase from dicyclohexylcarbodiimide-resistant mutants of Escherichia coli. 625 67

The photoautotrophic cyanobacterium Anacystis nidulans was used to investigate the membrane transport of branched-chain, neutral amino acids and its dependence on photosynthetic reactions. The uptake of alpha-amino [1-14C]isobutyric acid and L-[1-14C]leucine followed Michaelis, Menten kinetics and resulted in an energy-dependent accumulation. As in bacteria, different uptake systems for neutral amino acids were present: two DAG (D-alanine, aminoisobutyric acid, and glycine) systems responsible for uptake of alpha-amino [1-14C]isobutyric acid, and one LIV (leucine, isoleucine, and valine) system, responsible for uptake of leucine. The low-affinity DAG system seemed to be dependent on the presence of Na+ ions. Uptake was enhanced by white light and by monochromatic light of 630 nm. In far red light (717 nm) with and without nitrogen flushing, considerable uptake dependent on light intensity and inhibition by dibromothymoquinone and by high concentrations of KCN were observed. Therefore, the energy generated by photosystem I reactions only could perform this membrane transport. The proton translocator carbonylcyanide m-chlorophenylhydrazone and N,N-dicyclohexylcarbodiimide as an ATPase inhibitor reduced amino acid uptake to a high degree. A pH dependence of aminoisobutyric acid and leucine uptake was obvious, with a maximum at pH 6 to 7 and some at a pH as high as 9.5. At higher pH, increasing concentrations of Na+ K+ and also of triphenylmethylphosphonium ions inhibited the transport of aminoisobutyric acid. These findings are consistent with the assumption that ATP from photosynthetic reactions drives a membrane-bound proton-translocating ATPase producing a proton motive force, consisting at higher pH chiefly in a delta psi amount, which promotes a secondary active H+ or Na+/amino acid symport carrier.
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PMID:Amino acid uptake and energy coupling dependent on photosynthesis in Anacystis nidulans. 680 40

PotA protein, one of the components of the spermidine-preferential uptake system in Escherichia coli, was purified to homogeneity, and some of its properties were examined. PotA protein showed Mg(2+)-and SH-dependent ATPase activity. The specific activity was approximately 400 nmol/min/mg of protein and the Km value for ATP was 385 microM. The nature of the ATP binding site was explored by identification of the amino acid residue photoaffinity-labeled with 8-azido-ATP. It was found that 8-azido-ATP was attached to cysteine 26. In the spermidine transport-deficient mutant E. coli NH1596, valine 135 of PotA protein, which is located between two consensus amino acid sequences for nucleotide binding (50-57 and 168-173), was replaced by methionine (Kashiwagi, K., Miyamoto, S., Nukui, E., Kobayashi, H., and Igarashi, K. (1993) J. Biol. Chem. 268, 19358-19363). This mutated PotA protein could be labeled with 8-azido-ATP, but showed very low ATPase activity. To identify which cysteine is involved in the function of potA protein, cysteines 26, 54, and 276 were replaced by alanine, threonine, and alanine, respectively. Among the three mutated PotA proteins, the mutated PotA protein C54T only lost both ATPase and spermidine uptake activities. The results taken together indicate that the adenine portion of ATP interacts with a domain close to the NH2-terminal end of PotA protein, and active centers of ATP hydrolysis are located both within and between the two consensus amino acid sequences for nucleotide binding. Association of PotA protein with membranes was strengthened by the existence of channel forming PotB and PotC proteins. ATPase of PotA protein was inhibited by spermidine, suggesting that uptake inhibition by spermidine may function during this process.
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PMID:Spermidine-preferential uptake system in Escherichia coli. ATP hydrolysis by PotA protein and its association with membrane. 759 3

To define and differentiate primary and secondary RNA binding sites within the linear sequence of the rho protein, we investigated two mutant alleles, rho-115 and rhosuA1. They were first identified as defective in transcription termination in vivo, and later demonstrated to be defective in their interactions with RNA at the primary and secondary sites, respectively. Sequencing of rhosuA1 revealed a single lysine to glutamic acid residue change at position 352 (KE352), while rho-115 carries two mutations, glycine99 to valine (GV99) and a proline235 to histidine (PH235). Proteins carrying single mutations at each of these three positions were purified and their characteristics compared to the wild-type protein. We found both KE352 and GV99 to be defective in secondary-site RNA activation, with Km values for r(C)10 of 100 microM and approximately 650 microM, respectively, compared to the wild-type value of 4 microM. These observed secondary-site defects correlated with decreased helicase and ATPase activities, as well as a loss of transcription termination activity in vitro. By contrast, PH235 was very efficient at interacting with r(C)10 at the secondary site, with a measured Km of 0.5 microM, and displayed the characteristics of a hyperactive rho, as judged by its ATPase, helicase and termination capabilities. Our results show that mutations at three very different locations in the polypeptide can affect secondary-site activation by RNA, and that these interactions play a pivotal role in ATP hydrolysis, helicase activity and transcription termination.
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PMID:Analysis of E. coli rho factor: mutations affecting secondary-site interactions. 764 87

We analyzed the effect of the vacuolar H(+)-ATPase inhibitor bafilomycin A1 (bafA1) on the processing of beta-amyloid precursor protein (beta APP). In kidney 293 cells stably transfected with the wild-type beta APP cDNA, bafA1 caused a stabilization of mature beta APP and its 10-kDa COOH-terminal fragment. Moreover, it caused a 2-3-fold increase in secretion of soluble APP and amyloid-beta protein (A beta). Interestingly, bafA1 treatment of cells transfected with a mutant beta APP isoform that occurs in a Swedish kindred with familial Alzheimer's disease resulted in a decrease of A beta production and no increase of soluble APP secretion. Identical results were obtained when the effect of bafA1 was analyzed on fibroblasts derived from affected versus unaffected members of the Swedish family. These data demonstrate a differential effect of bafA1 on the production of A beta derived from wild-type or Swedish mutant beta APP. Radiosequencing of A beta derived from bafA1-treated cells expressing wild-type beta APP revealed a marked increase of A beta peptides starting at amino acids phenylalanine 4 and valine -3 and a relative decrease of A beta molecules beginning at the typical NH2 terminus of aspartate 1. Cells transfected with the Swedish mutation and treated with bafA1 did not produce these alternative A beta peptides, so that bafA1 treatment resulted in a decrease of A beta starting at aspartate 1. Our data indicate that multiple proteases are able to cleave A beta at or near its NH2 terminus. Inhibition of the protease cleaving at aspartate 1 by bafA1 and perhaps other similar agents can result in an increase of alternatively cleaved peptides.
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PMID:The vacuolar H(+)-ATPase inhibitor bafilomycin A1 differentially affects proteolytic processing of mutant and wild-type beta-amyloid precursor protein. 789 Jul 53

Sequences of the recA genes of the highly divergent thermopholic eubacteria Thermus aquaticus (and Thermus thermophilus), Thermotoga maritima, and Aquifex pyrophilus were determined from fragments derived by polymerase chain reaction (PCR) with degenerate primers and from inverse PCR products obtained using unique primers based on the fragment sequences. The source of the PCR products was verified by Southern hybridization. Complete PCR-derived recA genes were cloned into an expression vector regulated by a temperature-sensitive lambda-repressor, and independently derived clones expressing thermostable recA were selected. DNA sequences were verified to be authentic by direct cycle-sequencing of PCR products and/or sequencing of several clones. In contrast to Escherichia coli RecA protein, all the purified thermophilic RecA proteins exhibited single-stranded DNA-dependent ATPase activity optima above 70 degrees C. Phylogenetic analysis of RecA sequences suggested that the thermophilic RecA proteins were at least as different from one another as were Gram-positive organisms, mesophilic Gram-negative organisms, and cyanobacteria. In spite of substantial sequence divergence, interesting characteristics of the thermostable RecA proteins included increased valine content, common amino acid replacements at two highly conserved sites, and an increase in the calculated isoelectric point of approximately a full pH unit.
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PMID:Cloning, sequencing, and expression of RecA proteins from three distantly related thermophilic eubacteria. 792 98

The yeast Saccharomyces cerevisiae serves as an excellent model for the study of the structure and function of proteins. Numerous amino acid substitutions in the proteolipid subunit of yeast vacuolar H(+)-ATPase have been reported. Suppressed variants for several of the inactive mutants were selected after subjecting them to chemical or polymerase chain reaction mutagenesis and screening for second site suppressors. Suppressors for the mutation Gln90 to Lys change were intragenic and resulted from the changes: Ala14 to Val, Val74 to Ile, Ile89 to Leu, and Ile89 to Met. These residues are found on three different transmembrane segments but presumably at the same surface of the membrane. A new inactive proteolipid mutation was constructed by changing Val138 to Leu. This residue is situated in the middle of the fourth transmembrane segment, neighboring Glu137 which is the potential dicyclohexylcarbodiimide-binding site. The intragenic suppressor mutations for the above amino acid replacement resulted in changes of Val55 to Ala, Met59 to Val, or Ile130 to Thr. These residues are found in the second and fourth transmembrane segments, presumably on the same interface. It seems as if all those internal suppressor mutations compensate for the volume changes caused by the original displacement of the given amino acid. Five glycine residues, situated on the same face of the third transmembrane helix, were changed to valine and all these mutants were inactive. A suppressor mutation to one of those mutants (Gly101 to Val) was identified as substitution of Ile134 to Val. The structural and functional implications of these findings are discussed.
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PMID:Features of vacuolar H(+)-ATPase revealed by yeast suppressor mutants. 792 69

The vaccinia virus D6R open reading frame encodes the small subunit of the heterodimeric vaccinia virus early transcription factor (VETF) that activates transcription of early genes in vitro. VETF binds early gene promoters and has a DNA-dependent ATPase activity that is essential for activation of transcription. To examine the relationship between the structure and function of VETF, we have localized the mutations in two temperature-sensitive viruses whose lesions previously were mapped to the D6R gene. For both mutants, a single G-to-A nucleotide change that would alter protein coding potential was identified. In mutant E93, the codon for alanine 25 was changed to that of threonine, and in mutant S4 the codon for valine 278 was replaced with that for methionine. The molecular phenotype of each mutant was assessed by expressing mutant transcription factors in HeLa cells by using a vaccinia virus-T7 system and characterizing the proteins' activities in vitro. The A25T mutant activated transcription to a lesser extent than wild-type VETF, and the V278M mutant had no demonstrable transcription factor activity. Both mutant proteins were shown to be defective for promoter binding, accounting for their impairment in transcription activation. The functional defects for both mutants were observed at permissive as well as nonpermissive temperatures. The mutant proteins retained ATPase activity but required higher DNA concentrations to activate the ATPase. These results indicate that the small subunit of VETF is essential for its promoter binding activity and likely contacts the promoter DNA. Immunoblotting experiments showed that the virion particles from the two mutant viruses contained about half the VETF of wild-type virus, suggesting that promoter binding may contribute to packaging of VETF into the virion particle. RNA polymerase, mRNA capping enzyme, and nucleoside triphosphate phosphohydrolase I were found at similarly reduced levels in the virion, indicating that packaging of some virion core enzymes may be interdependent.
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PMID:Temperature-sensitive mutations in the gene encoding the small subunit of the vaccinia virus early transcription factor impair promoter binding, transcription activation, and packaging of multiple virion components. 813 39

The chaperone protein Hsc70 is an ATPase of unknown mechanism, although the crystal structure of the 44-kDa ATPase domain has been solved. This structure shows that the hydroxyl of threonine 204 is located close to the gamma-phosphate of ATP, in a position where it might be an intermediate phosphate acceptor in the hydrolysis reaction. We made two point mutations at residue 204 of Hsc70, threonine to valine (T204V) and threonine to glutamic acid (T204E). The wild-type ATPase domain had a Km for ATP of approximately 1 microM; the mutants had Km values of approximately 90 microM. The kcat values for the mutant proteins were also increased. After crystallization, the structures of the T204V and T204E proteins were solved and refined with data to 2.3- and 2.4-A resolution, respectively. The overall tertiary structure of the mutants showed little change from the wild type; however, significant changes were observed in the active site. Analysis of the structures suggested possible reasons for the changes in kinetic constants. Threonine 204 does not seem to be an obligatory intermediate phosphate acceptor in the hydrolysis reaction since the mutants retained appreciable ATPase activity.
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PMID:Threonine 204 of the chaperone protein Hsc70 influences the structure of the active site, but is not essential for ATP hydrolysis. 822 82

We evaluated the specific effects of acrolein on sulfhydryl status and plasma membrane-dependent functions of cultured pulmonary artery endothelial cells. Acrolein exposure caused a dose-dependent increase in lactate dehydrogenase (LDH) release and decreases in reduced glutathione (GSH) and protein sulfhydryl (P-SH) content, whereas oxidized glutathione (GSSG) content was not altered. Exposure to 4.5 microM, but not 1.5 or 3.0 microM, of acrolein caused significant (p < 0.05) LDH release. With increasing concentrations (25 microM) of acrolein, LDH release was increased to 66% (p < 0.001). Acrolein (3.0-25 microM) resulted in 36 to 100% reductions in GSH content, whereas reductions in P-SH content at these concentrations of acrolein ranged from 11 to 37%. Uptake of amino acids (cystine, glycine, and glutamic acid) and incorporation of valine into the protein fraction were significantly reduced in a dose-dependent fashion in acrolein (1.5-4.5 microM)-exposed cells. Reductions in cystine, glycine, and glutamic acid uptakes were maximal in cells exposed to 3 and 4.5 microM acrolein (p < 0.001). Similarly, maximum reductions (p < 0.001) in both uptake and incorporation of valine into the protein fraction were observed at 3.0 and 4.5 microM acrolein. Acrolein (1.5 microM) also resulted in significant loss of plasma membrane-specific Na+/K(+)-ATPase as well as plasma membrane P-SH content (p < 0.05 for both). When cells were treated with ouabain, reductions in amino acid uptake were observed, and this appeared to mimic the effect of acrolein exposure. When isolated plasma membranes were exposed to a known SH-alkylating agent, N-ethylmaleimide, losses of Na+/K(+)-ATPase and P-SH content were observed and were similar to the effects following exposure to acrolein. These results demonstrate that acrolein exposure results in alterations of plasma membrane-dependent transport in pulmonary artery endothelial cells, leading to reduced availability of precursor amino acids used in GSH and protein synthesis. This plasma membrane injury is accompanied by reductions in the GSH and P-SH contents of these cells. Loss of the plasma membrane P-SH appears to be associated with specific inactivation of Na+/K(+)-ATPase.
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PMID:Acrolein-induced injury to cultured pulmonary artery endothelial cells. 839 54


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