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)

Vanadate-sensitized photocleavage of the Ca2(+)-ATPase of rabbit sarcoplasmic reticulum was observed upon illumination of sarcoplasmic reticulum vesicles or the purified Ca2(+)-ATPase by ultraviolet light in the presence of 1 mM monovanadate or decavanadate. The site of the photocleavage is influenced by the Ca2+ concentration of the medium. When the [Ca2+] is maintained below 10 nM by EGTA, the vanadate-catalyzed photocleavage yields fragments of approximately equal to 87 and approximately equal to 22 kDa, while in the presence of 2-20 mM Ca, polypeptides of 71 and 38 kDa are obtained as the principal cleavage products. These observations indicate that the site of the vanadate-catalyzed photocleavage is altered by changes in the conformation of Ca2(+)-ATPase. Selective tryptic proteolysis, at Arg-505-Ala-506, combined with covalent labeling of Lys-515 by fluorescein 5'-isothiocyanate and with the use of anti-ATPase antibodies of defined specificity, permitted the tentative allocation of the sites of photocleavage to the A fragment near the T2 cleavage site in the absence of Ca2+, and to the B fragment between Lys-515 and Asp-659 in the presence of 2-20 mM Ca2+. The loss of ATPase activity during illumination is accelerated by calcium in the presence of vanadate. The vanadate-catalyzed photocleavage in the presence of Ca2+ is consistent with the existence of an ATPase-Ca2(+)-vanadate complex (Markus et al. (1989) Biochemistry 28, 793-799).
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PMID:Vanadate-catalyzed, conformationally specific photocleavage of the Ca2(+)-ATPase of sarcoplasmic reticulum. 213 45

The difference in susceptibility to audiogenic seizures (AGS) between C57BL/6J and DBA/2J inbred strains of mice is due to multiple genetic factors. AGS susceptibility was tested in 21-day-old mice from classical crosses, BXD recombinant inbred (RI) strains, a congenic DBA/2N.B6N-Ahb inbred strain and crosses between the BXD RI strains and DBA/2J. Analysis of these data reveals that the variation in AGS susceptibility between these two strains results from allelic differences at three or more loci. Most of the variation is due to allelic differences at two loci. The first, Asp-1 (formerly Ias), is a major gene located on chromosome 12, between Ah and D12 Nyul. The second, Asp-2 (formerly asp), is a minor gene located on chromosome 4, tightly linked to b. The negative correlation of brain stem Ca2(+)-ATPase activity and AGS susceptibility in the BXD RI strains suggests that the strain difference in Ca2(+)-ATPase activity is inherited as a polygenic trait and that Asp-1 and Asp-2 are linked to, or identical to, factors that influence Ca2(+)-ATPase activity.
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PMID:Mapping of two genes that influence susceptibility to audiogenic seizures in crosses of C57BL/6J and DBA/2J mice. 214 Dec 54

When a purified preparation of sarcoplasmic reticulum Ca2(+)-ATPase was labeled with 0.3 mM 7-chloro-4-nitrobenzo-2-oxa-1,3-diazole (NBD-Cl) in 1 mM AMPPNP and 1 mM CaCl2 at 25 degrees C and pH 7.0 for 60 min and then was treated with 10 mM dithiothreitol for 7 min, about 1 mol of NBD was incorporated per mol of the enzyme, and this inhibited the enzyme activity by 90 to 95%. The modified residue was identified as Cys-344 that is located near the phosphorylation site of the ATPase, Asp-351. The NBD-inhibition of enzyme activity could be reversed by treatment with membrane-acting agents such as C12E8, suggesting that Cys-344 is not directly involved in enzyme catalysis. A detailed study of partial reactions of ATP hydrolysis by the modified enzyme and associated changes in the fluorescence intensity of the incorporated NBD label revealed that a predominant effect of the NBD-modification was the inhibition of Ca2+ release from the ADP-sensitive phosphoenzyme intermediate and that two major fluorescent states of the enzyme alternated during ATP hydrolysis. The latter fluorescent data are consistent with the E1-E2 model of Ca2(+)-ATPase reaction.
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PMID:Does fluorescence of 4-nitrobenzo-2-oxa-1,3-diazole incorporated into sarcoplasmic reticulum ATPase monitor putative E1-E2 conformational transition? 214 2

The proteolipid subunit c of F1F0-type H(+)-transporting ATP synthases [ATP phosphohydrolase (H(+)-transporting), EC 3.6.1.34] contains a conserved Asp/Glu residue that is thought to function in H+ translocation. To test the importance of the position of this residue in the Escherichia coli enzyme, we used oligonucleotide-directed mutagenesis to move the carboxyl side chain from position 61 to position 58, 60, or 62. Mutant cells with these changes were incapable of growth via oxidative phosphorylation on succinate. An Asp-61----Glu mutant grew on succinate but at 50% the efficiency of wild type. Hence, even minor changes in the position of the carboxyl group can significantly reduce function. In a second approach, slow-growing revertants to an Asp-61----Gly mutant were isolated. In one such revertant, Ala-24 was changed to Asp, while the original Asp-61----Gly mutation remained unchanged. The Asp-24-Gly-61 double mutant grew on succinate at 60% the efficiency of wild type. Hence the essential carboxyl group of subunit c can function when anchored at either position 24 or position 61, and this supports the idea that these residues may neighbor each other when subunit c is folded in the membrane. The rate of ATP-driven H+ translocation by mutant membrane vesicles was estimated by the quenching of 9-amino-6-chloro-2-methoxyacridine fluorescence and corresponded to actual H+ pumping rates less than 25% that of wild type.
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PMID:The essential carboxyl group in subunit c of the F1F0 ATP synthase can be moved and H(+)-translocating function retained. 214 2

The sequences Thr-Gly-Glu-Ser184 and Asp-Gln-Ser178 and individual residues Asp149, Asp157, and Asp162 in the sarcoplasmic reticulum Ca2(+)-ATPase are highly conserved throughout the family of cation-transporting ATPases. Mutant Thr181----Ala, Gly182----Ala, Glu183----Ala, and Glu183----Gln, created by in vitro mutagenesis, were devoid of Ca2+ transport activity. None of these mutations, however, affected phosphorylation of the enzyme by ATP in the presence of Ca2+ or by inorganic phosphate in the absence of Ca2+, indicating that the high affinity Ca2(+)-binding sites and the nucleotide-binding sites were intact. In each of these mutants, the ADP-sensitive phosphoenzyme intermediate (E1P) decayed to the ADP-insensitive form (E2P) very slowly relative to the wild-type enzyme, whereas E2P decayed at a rate similar to that of the wild-type enzyme. Thus, the inability of the mutants to transport Ca2+ was accounted for by an apparent block of the transport reaction at the E1P to E2P conformational transition. These results suggest that Thr181, Gly182, and Glu183 play essential roles in the conformational change between E1P and E2P. Mutation of Ser184, Asp157, or Ser178 had little or no effect on either Ca2+ transport activity or expression. Mutations of Asp149, Asp162, and Gln177, however, were poorly expressed. Where expression could be measured, in mutations to Asp162 and Gln177, Ca2+ transport activity was essentially equivalent to that of the wild-type enzyme.
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PMID:Functional consequences of mutations of conserved amino acids in the beta-strand domain of the Ca2(+)-ATPase of sarcoplasmic reticulum. 214 58

At concentrations from 10 to 100 mM, inorganic phosphate and sulfate stimulate the activity of the H(+)-ATPase purified from the wild type Schizosaccharomyces pombe plasma membranes. Compared to the wild type ATPase, the stimulation by phosphate is more pronounced in the mutant pma1-1 (Gly-268----Asp) and is much reduced in the mutant pma1-2 (Lys-250----Thr) enzymes. In contrast, the inhibition by trifluoperazine is less pronounced in the pma1-1 mutant than in the wild type or pma1-2 mutant. The mutant pma1-2 ATPase activity is markedly stimulated by 10-20% dimethyl sulfoxide, which has a limited effect on the wild type and pma1-1 enzymes. These data indicate that the protein domain located in the beta-strand sector, including Lys-250 and Gly-268, is located in the active site and that its hydrophobic character influences the interactions of the yeast H(+)-ATPase with inorganic phosphate, as well as with the hydrophobic inhibitor trifluoperazine or the hydrophobic solvent dimethyl sulfoxide.
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PMID:Effects of phosphate and hydrophobic molecules on two mutations in the beta-strand sector of the H(+)-ATPase from the yeast plasma membrane. 214 85

Amino acids in three highly conserved segments of the Ca2(+)-ATPase. Asp-Pro-Pro-Arg604, Thr-Gly-Asp627, Thr-Gly-Asp703 as well as Asp707, have been proposed to participate in formation of the nucleotide binding site. We have tested this hypothesis by investigating the properties of mutants with alterations to amino acids within these segments. Most of the mutants were found to be defective in Ca2+ transport function. The inactive mutants could be separated into two classes on the basis of the kinetics of phosphoenzyme intermediate formation and decomposition. One group, Asp601----Asn, Pro603----Leu, Asp627----Glu, and Asp703----Asn, formed phosphoenzyme intermediates with ATP in the presence of Ca2+ and with inorganic phosphate only in the absence of Ca2+, indicating that both the high affinity Ca2+ binding sites and the nucleotide binding sites were intact. In each of these mutants, however, the ADP-sensitive phosphoenzyme intermediate (E1P) decayed to the ADP-insensitive phosphoenzyme intermediate very slowly, relative to the wild-type enzyme. Thus the inability of these mutants to transport Ca2+ was accounted for by an apparent block of the transport reaction at the E1P to E2P conformational transition. Another group, Asp601----Glu, Pro603----Gly, Asp707----Glu, and Asp707----Asn, did not form detectable phosphoenzyme intermediates from either ATP or Pi. Although we have demonstrated an effect on Ca2+ transport of mutations in each of the highly conserved regions predicted to be involved in ATP binding, we cannot yet define their roles in ATP-dependent Ca2+ transport.
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PMID:Functional consequences of alterations to amino acids located in the nucleotide binding domain of the Ca2(+)-ATPase of sarcoplasmic reticulum. 214 17

Point mutations of Asp-376 of the alpha-subunit of Torpedo californica Na+/K(+)-ATPase (the site of phosphorylation during the catalytic cycle) to Asn, Glu or Thr led to virtual abolishment of Na+/K(+)-ATPase activity and ouabain-binding capacity. Replacement of Lys-507 of the same subunit (the putative ATP-binding site) by Met resulted in decreases in Na+/K(+)-ATPase activity and ouabain-binding capacity. These results are in agreement with those reported for rabbit sarcoplasmic reticulum Ca2(+)-ATPase (Maruyama, K. and MacLennan, D.H. (1988) Proc. Natl. Acad. Sci. USA 85, 3314-3318).
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PMID:Site-directed mutagenesis of Asp-376, the catalytic phosphorylation site, and Lys-507, the putative ATP-binding site, of the alpha-subunit of Torpedo californica Na+/K(+)-ATPase. 215 58

Site-directed mutagenesis was used to identify residues responsible for the greater than 1,000-fold difference in ouabain sensitivity between the rat Na,K-ATPase alpha 1 and alpha 2 isoforms. A series of mutagenized cDNAs was constructed that replaced residues of the rat alpha 2 subunit with the corresponding residues from the rat alpha 1 subunit. These cDNAs were cloned into a mammalian episomal expression vector (EBOpLPP) and expressed in ouabain-sensitive primate cells. Either of two single substitutions introduced into the rat alpha 2 subunit cDNA (Leu-111----Arg or Asn-122----Asp) conferred partial resistance (approximately 10 microM ouabain) upon transformed cells. This resistance was intermediate between the levels conferred by the rat alpha 1 cDNA (approximately 500 microM ouabain) and the rat alpha 2 cDNA (approximately 0.2 microM ouabain). A double substitution of the rat alpha 2 cDNA (Leu-111----Arg and Asn-122----Asp) conferred a resistance level equivalent to that obtained with rat alpha 1. These results demonstrate that the residues responsible for isoform-specific differences in ouabain sensitivity are located at the end of the H1-H2 extracellular domain. The combination of site-directed mutagenesis and episomal expression provides a useful system for the selection and analysis of mutants.
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PMID:Ouabain-resistant mutants of the rat Na,K-ATPase alpha 2 isoform identified by using an episomal expression vector. 215 37

It has recently been shown that replacement of the border residues (Gln-111 and Asn-122) of the H1-H2 extracellular domain of the sheep Na,K-ATPase alpha subunit with the charged amino acids Arg and Asp generates a ouabain-resistant enzyme (Price, E. M. and Lingrel, J. B. (1988) Biochemistry 27, 8400-8408). In order to further study structure-function relationships in Na,K-ATPase, six additional mutations have been made at these border positions. Two of these mutants were single amino acid substitutions (Gln-111 to Arg or Asn-122 to Asp). These mutations change one or the other H1-H2 border residue to a charged amino acid. The remaining substitutions were double mutants in which both of the H1-H2 border residues were simultaneously changed to charged amino acids. Changes were made which introduced either positively charged amino acids (Lys at positions 111 and 122), negatively charged amino acids (Glu at positions 111 and 122) or oppositely charged amino acids (Lys at position 111 and Glu at 122; Asp at position 111 and Arg at 122) at the borders of the H1-H2 extracellular domain. HeLa cells transfected with any of these sheep Na,K-ATPase alpha subunit mutants were able to grow in concentrations of ouabain that were toxic to untransfected cells or cells transfected with the wild type sheep alpha subunit. Crude membranes isolated from the transfectants were analyzed for ouabain inhibitable Na,K-ATPase activity. All of the transfectants contained a relatively ouabain-resistant component of enzyme activity, with the ouabain I50 values ranging from 4 x 10(-3) M to 1 x 10(-6) M. The most resistant enzyme was the double mutant that contained Asp at position 111 and Arg at 122, whereas the least resistant were the enzymes containing the single amino acid substitutions. There was no correlation between the type of charged amino acid present at the border position and the degree of ouabain resistance. These data demonstrate the functional importance, in terms of ouabain binding, of the border positions of the H1-H2 extracellular domain of the Na,K-ATPase alpha subunit.
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PMID:Structure-function studies of Na,K-ATPase. Site-directed mutagenesis of the border residues from the H1-H2 extracellular domain of the alpha subunit. 215 5


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