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)

Complementary DNA to pig gastric mRNA encoding (H+ + K+)-ATPase was cloned, and its amino acid sequence was deduced from the nucleotide sequence. The enzyme contained 1034 amino acid residues (Mr. 114,285) including the initiation methionine. The sequence of pig (H+ + K+)-ATPase was highly homologous with that of the corresponding enzyme from rat, but had high degree of synonymous codon changes. Potential sites of phosphorylation by cAMP-dependent protein kinase and N-linked glycosylation sites were identified. The amino terminal region contained a lysine-rich sequence similar to that of the alpha subunit of (Na+ + K+)-ATPase, although a cluster of glycine residues was inserted into the sequence of the (H+ + K+)-ATPase. As the pig enzyme is advantageous for biochemical studies, the information of the primary structure is useful for further detailed studies.
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PMID:cDNA cloning and sequence determination of pig gastric (H+ + K+)-ATPase. 284 18

cDNA fragments coding for the alpha and beta subunits of the Na,K-ATPase were separately ligated into the yeast expression vector YEp1PT in both the sense (YEpNKA(+)) and anti-sense (YEpNKA(-)) orientations with respect to the promoter. The recombinant plasmids were introduced into Saccharomyces cerevisiae strain UT4 by transformation. Total RNA from the transformed strains was isolated and analyzed by Northern hybridization. The resulting autoradiogram revealed strong signals indicative of a high level of transcriptional expression of both subunits in both orientations of the cDNA. 35S-Methionine labeled extracts were immunoprecipitated with antibodies specific for the beta subunit. A beta subunit translation product was produced from YEp beta NKA(+) but not from YEp beta NKA(-). Experiments to detect an alpha specific translation product are in progress.
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PMID:Development of a heterologous gene expression system for the Na,K-ATPase subunits in the yeast Saccharomyces cerevisiae. 285 34

The saturable, carrier-mediated system capable of the brain-to-blood transport of small peptides with an N-terminal tyrosine was characterized. The rate of disappearance of intraventricularly injected iodinated peptide in the presence or absence of the inhibitor being tested was determined from formulas based on the residual radioactivity in the brains of mice after decapitation. The injection of 100 nmol/mouse of unlabeled N-Tyr-MIF-1 (TMIF) increased the half-time disappearance of 125I-TMIF (ITMIF) in the central nervous system (CNS) from 14.1 to 88.7 min (P less than 0.00005). Technetium, a substance transported out of the brain by the same system that transports iodine, was used as a control; the half-time disappearance of technetium pertechnetate was unaffected by unlabeled TMIF. With two related but distinct techniques, the maximum transport rate out of the CNS (Vmax) for TMIF was 0.266 nmol X g of brain per min (method 1) and 0.297 nmol X g-1 X min-1 (method 2), while the amount of unlabeled material needed to achieve 50% of Vmax (Km) was 15.2 nmol/g (method 1) and 15.1 nmol/g (method 2). The lack of effect of the tyrosinated fragments of TMIF as inhibitors indicates that TMIF is being transported in intact form. The Vmax for methionine enkephalin determined with labeled and unlabeled methionine enkephalin was 0.630 nmol X g-1 X min-1 and the Km was 24.95 nmol/g. Studies with the metabolic modulators furosemide, acetozolamide, reserpine, ouabain, and theophylline suggest that the system is sodium dependent and probably independent of ATPase.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Carrier-mediated transport of enkephalins and N-Tyr-MIF-1 across blood-brain barrier. 287 44

A single-gene nuclear mutant has been selected from the yeast Schizosaccharomyces pombe for growth resistance to Dio-9, a plasma membrane H+-ATPase inhibitor. From this mutant, called pma1, an ATPase activity has been purified. It contains a Mr = 100,000 major polypeptide which is phosphorylated by [gamma-32P] ATP. Proton pumping is not impaired since the isolated mutant ATPase is able, in reconstituted proteoliposomes, to quench the fluorescence of the delta pH probe 9-amino-6-chloro-2-methoxy acridine. The isolated mutant ATPase is sensitive to Dio-9 as well as to seven other plasma membrane H+-ATPase inhibitors. The mutant H+-ATPase activity tested in vitro is, however, insensitive to vanadate. Its Km for MgATP is modified and its ATPase specific activity is decreased. The pma1 mutation decreases the rate of extracellular acidification induced by glucose when cells are incubated at pH 4.5 under nongrowing conditions. During growth, the intracellular mutant pH is more acid than the wild type one. The derepression by ammonia starvation of methionine transport is decreased in the mutant. The growth rate of pma1 mutants is reduced in minimal medium compared to rich medium, especially when combined to an auxotrophic mutation. It is concluded that the H+-ATPase activity from yeast plasma membranes controls the intracellular pH as well as the derepression of amino acid, purine, and pyrimidine uptakes. The pma1 mutation modifies several transport properties of the cells including those responsible for the uptake of Dio-9 and other inhibitors (Ulaszewski, S., Coddington, A., and Goffeau, A. (1986) Curr. Genet. 10, 359-364).
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PMID:A single mutation confers vanadate resistance to the plasma membrane H+-ATPase from the yeast Schizosaccharomyces pombe. 287 25

Mutant genes for the gamma subunit of H+-translocating ATPase (H+-ATPase) were cloned from eight different strains of Escherichia coli isolated in this laboratory. Determination of their nucleotide sequences revealed that they are amber nonsense mutations: a Gln codon at position 15, 158, 227, 262, and 270, respectively, was replaced by a termination codon in these strains. As terminal Met is missing in the gamma subunit, these results indicate that these strains are capable of synthesizing fragments of gamma subunits of 13, 156, 225, 260, and 268 amino acid residues, respectively. Studies on the properties of membranes of these strains suggested the importance of the region between Gln 269 and the carboxyl terminus (residue 286) for forming a stable F1 complex with ATPase activity and the region between Gln 226 and Gln 261 for normal interaction of F1 with F0. The sequence from Gln 261 to Gln 269 also seemed to be important for stability of F1 assembly on the membranes. The high frequency of the nonsense mutations suggested that the number of essential residues is limited in this subunit. Comparison of the homologies of the amino acid sequences of the gamma subunits from four different sources confirmed this notion: 19% of amino acid residues are identically conserved in these four strains, and the conserved regions are the amino terminal and carboxyl terminal regions.
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PMID:Escherichia coli H+-ATPase: loss of the carboxyl terminal region of the gamma subunit causes defective assembly of the F1 portion. 287 11

Six mutant uncD alleles, affecting essential residues of the beta-subunit of Escherichia coli proton-ATPase, have been identified by intragenic complementation mapping, cloning, and DNA sequencing. Five of the mutations impair catalysis but do not cause structural perturbation of F1-ATPase. The amino acid substitutions found were as follows: uncD412, Gly-142----Ser; uncD430 and uncD431, both Arg-246----Cys; uncD478, Ser-174----Phe; and uncD484, Met-209----Ile. Kinetic characteristics of each corresponding mutant F1-ATPase are described or reviewed. In each case, the major determinant of impaired catalysis appears to be an attenuation of positive catalytic site cooperativity. Additionally, each mutation affects intrinsic properties of the catalytic site, including affinity for ATP, the ratio between unisite-bound substrate and products, and the rate of release of product inorganic phosphate under unisite ATP hydrolysis conditions. These effects are discussed in terms of a structural model of the catalytic nucleotide-binding domain of beta-subunit proposed recently (Duncan, T.M., Parsonage, D., and Senior, A.E. (1986) FEBS Lett. 208, 1-6). Each of the mutations lies within that domain. The uncD409 allele abolishes normal assembly of F1-ATPase. The amino acid substitution is Gly-214----Arg, which is suggested to affect a beta-turn connecting a beta-strand and an alpha-helix in the predicted nucleotide-binding domain of the beta-subunit.
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PMID:The defective proton-ATPase of uncD mutants of Escherichia coli. Identification by DNA sequencing of residues in the beta-subunit which are essential for catalysis or normal assembly. 288 84

Photolabeling of nucleotide binding sites in nucleotide-depleted mitochondrial F1 has been explored with 2-azido [alpha-32P]adenosine diphosphate (2-N3[alpha-32P] ADP). Control experiments carried out in the absence of photoirradiation in a Mg2+-supplemented medium indicated the presence of one high affinity binding site and five lower affinity binding sites per F1. Similar titration curves were obtained with [3H]ADP and the photoprobe 3'-arylazido-[3H]butyryl ADP [( 3H]NAP4-ADP). Photolabeling of nucleotide-depleted F1 with 2-N3[alpha-32P]ADP resulted in ATPase inactivation, half inactivation corresponding to 0.6-0.7 mol of photoprobe covalently bound per mol F1. Only the beta subunit was photolabeled, even under conditions of high loading with 2-N3[alpha-32P]ADP. The identification of the sequences labeled with the photoprobe was achieved by chemical cleavage with cyanogen bromide and enzymatic cleavage by trypsin. Under conditions of low loading with 2-N3[alpha-32P]ADP, resulting in photolabeling of only one vacant site in F1, covalently bound radioactivity was located in a peptide fragment of the beta subunit spanning Pro-320-Met-358 identical to the fragment photolabeled in native F1 (Garin, J., Boulay, F., Issartel, J.-P., Lunardi, J., and Vignais, P. V. (1986) Biochemistry 25, 4431-4437). With a heavier load of photoprobe, leading to nearly 4 mol of photoprobe covalently bound per mol F1, an additional region of the beta subunit was specifically labeled, corresponding to a sequence extending from Gly-72 to Arg-83. The isolated beta subunit also displayed two binding sites for 2-N3-[alpha-32P]ADP. When F1 was first photolabeled with a low concentration of NAP4-ADP, leading to the covalent binding of 1.5 mol of NAP4-ADP/mol F1, with the bound NAP4-ADP distributed equally between the alpha and beta subunits, a subsequent photoirradiation in the presence of 2-N3[alpha-32P]ADP resulted in covalent binding of the 2-N3[alpha-32P]ADP to both alpha and beta subunits. It is concluded that each beta subunit in mitochondrial F1 contains two nucleotide binding regions, one of which belongs to the beta subunit per se, and the other to a subsite shared with a subsite located on a juxtaposed alpha subunit. Depending on the experimental conditions, the subsite located on the alpha subunit is either accessible or masked. Unmasking of the subsite in the three alpha subunits of mitochondrial F1 appears to proceed by a concerted mechanism.
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PMID:Mapping of nucleotide-depleted mitochondrial F1-ATPase with 2-azido-[alpha-32P]adenosine diphosphate. Evidence for two nucleotide binding sites in the beta subunit. 288 35

The effect of various opioids on Na+, K+ -ATPase partially purified from rat heart was examined. Dynorphin-A (1-13), dynorphin-A (1-17) and ethylketocyclazocine (EKC), which are k-type opiate agonists, markedly inhibited the enzyme activity in a dose-dependent manner; IC50 values were 12 microM, 21 microM and 0.38 mM, respectively. Morphine (mu-type agonist), methionine- and leucine-enkephalin (delta-type agonist) at the concentration of 1 mM did not affect the enzyme activity. The effect of dynorphin-A (1-13) and EKC was not antagonized by naloxone. Dynorphin-A (1-13) mainly decreased Vmax value without the change of Km value in the activation of Na+, K+-ATPase by ATP, Na+ and K+. Dynorphin-A(1-13) inhibited the partial reactions of Na+, K+-ATPase at the different degree of the potency; the inhibition of K+-stimulated phosphatase was greater than that of Na+-dependent phosphorylation. The present study suggests that dynorphin-A and EKC have an effect on cardiovascular system which is mediated by the inhibition of Na+, K+-ATPase in the heart.
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PMID:Inhibition of cardiac Na+, K+-ATPase activity by dynorphin-A and ethylketocyclazocine. 289 34

The plasma membrane H+-ATPase of Neurospora is a 100-kDa integral membrane protein which appears, on the basis of hydropathy analysis of its amino acid sequence, to span the lipid bilayer at least eight times. To investigate the assembly and processing of the ATPase, a full-length cDNA has been constructed for use in in vitro transcription and translation experiments. Comparison of three different forms of the ATPase (nascent protein, nascent protein cotranslationally inserted into membranes, and mature protein) revealed no difference in electrophoretic mobility. Furthermore, the nascent and mature forms gave identical peptide patterns after partial proteolysis with Staphylococcus aureus V8 protease, suggesting that the ATPase does not contain an NH2-terminal signal peptide which is cleaved upon membrane insertion. Consistent with this interpretation, the NH2-terminal peptide has been purified from a tryptic digest of the ATPase and found to lack only the initiator methionine residue; the penultimate alanine is acetylated based on analysis by fast atom bombardment mass spectroscopy. Although the ATPase contains one potential site of N-linked glycosylation, its electrophoretic mobility was unchanged following digestion with endoglycosidase H and it did not incorporate [3H]mannose or bind concanavalin A. Thus, the Neurospora plasma membrane-ATPase appears to undergo minimal post-translational processing, and its membrane insertion is probably mediated by internal sequences.
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PMID:Biosynthesis of the plasma membrane H+-ATPase of Neurospora crassa. 290 84

Six chromosomal uncF mutants of Escherichia coli defective in the b subunit of H+-ATPase (156 amino acid residues) were identified (KF92, Met-1----Val; KF164, Gln-64----end; KF61 and KF144, Gln-104----end; KF138, Gln-106----end; and KF79, Gln-123----end). The membranes of all these mutants had low ATPase activities (less than 5% of that of the wild type), and no functional H+ pathway, although the truncated b subunits were integrated into these membranes. These findings suggest that about 30 carboxyl-terminal amino acid residues of the b subunit are essential for formation of the F1-binding site and H+ pathway. For examination of the role(s) of the carboxyl-terminal region(s) or residue(s) of the b subunit, recombinant plasmids carrying truncated uncF genes of various lengths were constructed by in vitro muta-genesis and introduced into a recA1 derivative of strain KF92 (Met-1----Val). Analyses of the membranes from the resulting strains demonstrated that almost the entire carboxyl-terminal region of the b subunit is necessary for formation of functional Fo, since loss of the carboxyl-terminal residue resulted in significant reduction of both F1 binding and H+ translocation, and loss of two or more residues abolished both activities completely.
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PMID:Fo portion of Escherichia coli H+-ATPase. Carboxyl-terminal region of the b subunit is essential for assembly of functional Fo. 290 50


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