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

ATPase activity of the coupling factor 1, CF1, isolated from spinach chloroplasts, was enhanced by reduction with dithiothreitol. Reduced thioredoxins from spinach chloroplasts, Escherichia coli and human lymphocytes replaced dithiothreitol as reductant and activator of the ATPase. CF1 must be in an oxidized activated state to be further activated by reduced thioredoxin. This state was obtained either by heating CF1 or removing the inhibitory intrinsic epsilon subunit from CF1. Efficiency and primary structure of the different thioredoxins were compared. The progressive addition of KCl during ATPase activation by reduced thioredoxin increases then decreases this process. We proposed that three basic amino acids corresponding to arginine 73 and lysines 82 and 96 in Escherichia coli thioredoxin play an important role in the anchorage of the thioredoxin to the negatively charged surface of the CF1 and are involved in the dual effect of KCl. The variations in the screening effect of the negative charges of the CF1 surface by K+ ions can indeed explain the changes in the anchorage of these 3 basic amino acids with concomitant variation in ATPase activity. Human thioredoxin must be 10 times more concentrated than Escherichia coli or spinach chloroplast thioredoxin to exhibit the same activation effect on the ATPase. This fact was related to the properties of a sequence equivalent to the part from amino acid 59 to 72 in Escherichia coli thioredoxin. This part which joins the two lobes of the thioredoxin is more hydrophilic and more negatively charged in human thioredoxin than in Escherichia coli or spinach chloroplast thioredoxin. Although ATPase activation was obtained at a very low concentration of the reduced spinach chloroplast thioredoxin, the thioredoxin formed only a loose complex with CF1.
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PMID:Induction by different thioredoxins of ATPase activity in coupling factor 1 from spinach chloroplasts. 214 Feb 77

Previous studies have shown that the initial complex formed when ADP binds to nucleotide-depleted F1-ATPase is transformed with a half time of 2 to 3 min to form with a much lower rate of ADP release. The ADP binding results in a strong inhibition of ATPase activity. The present paper reports appraisal of where the inhibitory ADP binds by use of the photoreactive ADP analog, 2-N3-ADP. In presence of Mg2+ the 2-N3-ADP like ADP induces reversible inhibition of nucleotide-depleted F1 (ndF1) with a Kd of about 10 nM. Photoirradiation of the inactive 2-N3-[beta-32P]ADP-ndF1 complex results in labeling of only the beta-subunit. The major labeled peptide isolated from a trypic digest consists of residues from Ala-338 to Arg-356, with Tyr-345 as the site of labeling. This identifies the site of the inhibitory ADP binding as one of the catalytic sites of the enzyme.
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PMID:The ADP that binds tightly to nucleotide-depleted mitochondrial F1-ATPase and inhibits catalysis is bound at a catalytic site. 214 75

Europium luminescence from europium bound to sarcoplasmic reticulum (Ca2+ + Mg2+)-ATPase indicates that there are two high affinity calcium binding sites. Furthermore, the two calcium ions at the binding sites are highly coordinated by the protein as the number of H2O molecules surrounding the Ca2+ ions are 3 and 0.5. In the presence of ATP, calcium ions are occluded even further down to 2 and zero H2O molecules, respectively. The Ca2+ - Ca2+ intersite distance is estimated to be 8-9 A and the average distance from the Ca2+ sites to CrATP is about 18 A. Digestion of the (Ca2+ + Mg2+)-ATPase at the T2 site (Arg 198) causes uncoupling of Ca2(+)-transport from ATPase activity while calcium occlusion due to E1-P formation remains unchanged. Further tryptic digestion beyond T2 and in the presence of ATP diminishes Ca2+ occlusion to zero while 50% of the ATPase hydrolytic activity remains. Tryptic digestion beyond T2 and in the absence of ATP diminishes ATPase hydrolytic activity to 50% of normal while Ca2+ occlusion remains intact. These data are consistent with a mechanism in which the functional enzyme must be in the dimeric form for occlusion and calcium uptake to occur, but each monomer can hydrolyze ATP.
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PMID:Tertiary structure and energy coupling in Ca2(+)-pump system. 214 85

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

We have previously shown that melittin, a bee venom peptide, potently inhibited the catalytic and transport functions of rabbit gastric (H+ + K+)ATPase. A radioactive photoaffinity analog of melittin, ([125I]azidosalicylyl melittin), labeled the (H+ + K+)ATPase. These results suggested that melittin exerted inhibitory effects through direct interaction with the (H+ + K+)ATPase. In this study we attempt to define the melittin-binding domain of the (H+ + K+)ATPase using conformation-dependent proteolytic fragmentation of [125I]azidosalicylyl melittin-labeled hog gastric (H+ + K+)ATPase. In the presence of KCl (E2 form) the 95,000-Da [125I]-azidosalicylyl melittin-labeled (H+ + K+)ATPase was cleaved by trypsin to a 40,000-Da NH2-terminal tryptic fragment and a 56,000-Da COOH-terminal fragment through cleavage at Arg 454 of the (H+ + K+)ATPase. The 40,000-Da fragment was labeled by [125I]-azidosalicylyl melittin. The 56,000-Da fragment was not labeled. When unmodified (H+ + K+)ATPase was trypsinized in the presence of KCl, and the fragments were then reacted with [125I]azidosalicylyl melittin, similar tryptic fragmentation results were obtained. In the absence of KCl (E1 form), the 56,000- and 40,000-Da fragments did not accumulate. Chymotryptic hydrolysis of [125I]azidosalicylyl melittin-labeled (H+ + K+)-ATPase was very slow in the presence of KCl (E2 form). In the absence of KCl (E1 form), chymotryptic hydrolysis was more rapid, with accumulation of a major 42,000-Da fragment which was radiolabeled. The melittin-binding region on the (H+ + K+)ATPase is N-terminal to Arg 454 of the (H+ + K+)ATPase. This region is known to contain the aspartyl phosphate residue (Asp 385), the site of phosphoenzyme formation on the (H+ + K+)ATPase. Melittin is also known to bind to calmodulin and other proteins. Another known calmodulin-binding peptide with a different sequence but similar structure, Trp-3, (Leu-Lys-Trp-Lys-Lys-Leu-Leu-Lys-Leu-Leu-Lys-Lys-Leu-Leu-Lys-Leu-Gly) also inhibited the (H+ + K+)ATPase and label incorporation by [125I]azidosalicylyl melittin. These Trp-3 results suggested that the (H+ + K+)ATPase contains a peptide-binding domain which is similar to the peptide-binding domains found on other melittin-binding proteins.
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PMID:[125I]azidosalicylyl melittin binding domains: evidence for a polypeptide receptor on the gastric (H+ + K+)ATPase. 215 80

The actin-activated Mg2(+)-ATPase activity of myosin II from Acanthamoeba castellanii is regulated by phosphorylation of 3 serines in its 29-residue, nonhelical, COOH-terminal tailpiece, i.e., serines-1489, -1494, and -1499 or, in reverse order, residues 11, 16, and 21 from the COOH terminus. To investigate the essential requirements for regulation, myosin II filaments in the presence of F-actin were digested by arginine-specific submaxillary gland protease. Two-dimensional peptide mapping of purified, cleaved myosin II showed that the two most terminal phosphorylation sites, serines-1494 and -1499, had been removed. Cleaved dephosphorylated myosin II retained full actin-activated Mg2(+)-ATPase activity (with no change in Vmax or Kapp) and the ability to form filaments similar to those of the native enzyme. However, higher Mg2+ concentrations were required for both filament formation and maximal ATPase activity. The one remaining regulatory serine in the cleaved myosin II was phosphorylatable by myosin II heavy-chain kinase, and phosphorylation inactivated the actin-activated Mg2(+)-ATPase activity, as in the case of the native myosin II. Also as in the case of the native myosin II, phosphorylated cleaved myosin II inhibited the actin-activated Mg2(+)-ATPase activity of dephosphorylated cleaved myosin II when the two were copolymerized. These results suggest that at least 18 of the 29 residues in the nonhelical tailpiece of the heavy chain are not required for either actin-activated Mg2(+)-ATPase activity or filament formation and that phosphorylation of Ser-1489 is sufficient to regulate the actin-activated Mg2(+)-ATPase activity of myosin II.
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PMID:Functional consequences of the proteolytic removal of regulatory serines from the nonhelical tailpiece of Acanthamoeba myosin II. 216 Feb 67

Tryptic digestion of pig renal Na/K-ATPase in the presence of Rb and absence of Ca ions removes about half of the protein but leaves a stable 19-kDa membrane-embedded fragment derived from the alpha chain, a largely intact beta chain, and essentially normal Rb- and Na-occlusion capacity. Subsequent digestion with trypsin in the presence of Ca or absence of Rb ions leads to rapid loss of the 19-kDa fragment and a parallel loss of Rb occlusion, demonstrating that the fragment is essential for occlusion. The N-terminal sequence of the 19-kDa fragment is Asn-Pro-Lys-Thr-Asp-Lys-Leu-Val-Asn-Glu-Arg-Leu-Ile-Ser-Met-Ala, beginning at residue 830 and extending toward the C terminus. Membranes containing the 19-kDa fragment have the following functional properties. (i) ATP-dependent functions are absent. (ii) The apparent affinity for occluding Rb is unchanged, the affinity for Na is lower than in the control enzyme, and activation is now strongly sigmoidal rather than hyperbolic. (iii) Membranes containing the 19-kDa fragment can be reconstituted into phospholipid vesicles and sustain slow Rb-Rb exchange. Thus the transport pathway is retained. We conclude that cation occlusion sites and the transport pathway within transmembrane segments are quite separate from the ATP binding site, located on the cytoplasmic domain of the alpha chain. Interactions between cation and ATP sites, the heart of active transport, must be indirect--mediated, presumably, by conformational changes of the protein.
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PMID:A 19-kDa C-terminal tryptic fragment of the alpha chain of Na/K-ATPase is essential for occlusion and transport of cations. 216 48

Phosphorylation of a single threonine (myosin IA) or serine (myosins IB and IC) in the heavy chains of the Acanthamoeba myosin I isozymes is required for expression of their actin-activated Mg2(+)-ATPase activities. We now report that the synthetic peptide Gly-Arg-Gly-Arg-Ser-Ser-Val-Tyr-Ser, which corresponds to the phosphorylated region of Acanthamoeba myosin IC, is a good substrate for myosin I heavy chain kinase: Km = 54 microM, and Vmax = 15 mumols/min.mg. The same serine is phosphorylated as in the native substrate (residue 6 in the above sequence), and kinase activity with the synthetic peptide as substrate is also stimulated by phosphatidylserine-enhanced autophosphorylation of the kinase. These results indicate that all of the essential sequence determinants of kinase specificity are contained within this 9-residue peptide. With the peptide as substrate, we found that another acidic phospholipid, phosphatidylinositol, also enhances autophosphorylation of the kinase whereas the neutral phospholipids phosphatidylcholine and phosphatidylethanolamine do not. By comparing the Km and Vmax values for a series of synthetic peptide substrates, we established that 1 basic amino acid is essential on the NH2-terminal side of the phosphorylation site, and two are preferable, and that a tyrosine is essential 2 residues away on the COOH-terminal side. There is a slight preference for arginines over lysines. All of these local sequence specificity determinants are present in the three native substrates, Acanthamoeba myosins IA, IB, and IC, and in two Dictyostelium myosin I isozymes that are putative substrates for the kinase. Similar sequences do not occur in the myosins I from intestinal brush border, which is not a substrate for the Acanthamoeba kinase.
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PMID:Substrate specificity of Acanthamoeba myosin I heavy chain kinase as determined with synthetic peptides. 216 81


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