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)

Phospholamban inhibits the Ca(2+)-ATPase of cardiac sarcoplasmic reticulum by lowering its affinity for Ca2+. In earlier studies (Toyofuku, T., Kazimierz, K., Tada, M., and MacLennan, D. H. (1993) J. Biol. Chem. 268, 2809-2815), parts of the phosphorylation and nucleotide binding/hinge domains of the Ca(2+)-ATPase were shown to be essential for phospholamban interaction. In order to identify the sites in phospholamban which interact with the Ca(2+)-ATPase, a series of mutants containing amino acid replacements in the cytoplasmic and transmembrane regions of phospholamban were co-expressed with the cardiac/slow-twitch muscle Ca(2+)-ATPase isozyme, SERCA2a, in HEK-293 cells. Mutation of residues in the cytoplasmic 1A domain of phospholamban, including positively charged residues, Lys3, Arg9, Arg13, and Arg14, negatively charged residue, Glu2, hydrophobic residues, Val4, Leu7, Ala11, Ile12, Ala15, and Ile18, and phosphorylation site residues, Ser16 and Thr17, resulted in loss of the inhibitory effect of phospholamban on Ca2+ transport by the Ca(2+)-ATPase. By contrast, mutation of Met1, Gln5, Tyr6, Thr8, Ser10, Glu19, or Met20 or of residues in the cytoplasmic 1B domain (Pro21 to Asn30) and of Cys41 in the transmembrane domain (Leu31 to Leu52) did not diminish the inhibitory effects of phospholamban on Ca2+ transport. These results suggest that a region essential for functional association of phospholamban with the Ca(2+)-ATPase lies in the cytoplasmic 1A domain of phospholamban.
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PMID:Amino acids Glu2 to Ile18 in the cytoplasmic domain of phospholamban are essential for functional association with the Ca(2+)-ATPase of sarcoplasmic reticulum. 790 83

Central to the chaperone function of Hsp70 stress proteins including Escherichia coli DnaK is the ability of Hsp70 to bind unfolded protein substrates in an ATP-dependent manner. Mg2+/ATP dissociates bound substrates and, furthermore, substrate binding stimulates the ATPase of Hsp70. This coupling is proposed to require a glutamate residue, E175 of bovine Hsc70, that is entirely conserved within the Hsp70 family, as it contacts bound Mg2+/ATP and is part of a hinge required for a postulated ATP-dependent opening/closing movement of the nucleotide binding cleft which then triggers substrate release. We analyzed the effects of dnaK mutations which alter the corresponding glutamate-171 of DnaK to alanine, leucine or lysine. In vivo, the mutated dnaK alleles failed to complement the delta dnaK52 mutation and were dominant negative in dnaK+ cells. In vitro, all three mutant DnaK proteins were inactive in known DnaK-dependent reactions, including refolding of denatured luciferase and initiation of lambda DNA replication. The mutant proteins retained ATPase activity, as well as the capacity to bind peptide substrates. The intrinsic ATPase activities of the mutant proteins, however, did exhibit increased Km and Vmax values. More importantly, these mutant proteins showed no stimulation of ATPase activity by substrates and no substrate dissociation by Mg2+/ATP. Thus, glutamate-171 is required for coupling of ATPase activity with substrate binding, and this coupling is essential for the chaperone function of DnaK.
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PMID:The chaperone function of DnaK requires the coupling of ATPase activity with substrate binding through residue E171. 790 76

We studied the contraction characteristics and Mg-ATPase activity of glycerinated rabbit psoas muscle fibers in the presence and absence of polyclonal antibody directed against the subfragment-2 (S-2) region of myosin, to give information about the role of myosin hinge region in muscle contraction. The antibody was kindly supplied to us from Professor Harrington's laboratory. The antibody-induced decrease of Ca(2+)-activated isometric force development was always accompanied by a parallel decrease of muscle fiber stiffness, so that the stiffness versus force relation remained the same by the antibody treatment. Force-velocity curves, obtained by applying ramp decreases in load from steady isometric force to zero, indicated that the antibody had no effect on the maximum shortening velocity and the shape of the force-velocity curve. Simultaneous measurements of Mg-ATPase activity and Ca(2+)-activated isometric force showed that Mg-ATPase activity of the fibers remained unchanged despite the antibody-induced decrease of isometric force even to zero. These results indicate that, if the antibody attaches to the S-2 region of myosin molecules, their heads still hydrolyze ATP without contributing to both muscle force generation and muscle fiber stiffness.
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PMID:Essential role of myosin S-2 region in muscle contraction. 810 73

The Ca(2+)-ATPase of skeletal muscle sarcoplasmic reticulum can be labelled at Cys-670 and Cys-674 with 5-[[2-[(iodoacetyl) amino]ethyl]amino]naphthalene-1-sulphonic acid (IAEDANS). Resonance energy transfer has been used to measure the distance between Cys-670/Cys-674 and Glu-439 labelled with 5-(bromomethyl)fluorescein as 40 A. The height of Cys-670/Cys-674 above the phospholipid/water interface has been measured by resonance energy transfer between IAEDANS-labelled ATPase and fluorescein-labelled phosphatidylethanolamine as 54 A. This locates the hinge region of the ATPase close to the mouth of the pore observed in the cytoplasmic region of the ATPase in electron micrographs. No significant changes in these distances can be detected by resonance energy transfer on binding Ca2+ or vanadate. The height of the IAEDANS label above the phospholipid/water interface is the same for bilayers of dimyristoleoylphosphatidylcholine and dioleoylphosphatidylcholine. Conformation changes on the Ca(2+)-ATPase appear to be localised to small regions of the ATPase.
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PMID:Localization of the hinge region of the Ca(2+)-ATPase of sarcoplasmic reticulum using resonance energy transfer. 820 50

The amino acid sequences of several P-type ATPases share regions of high similarity. The functions of some of these regions, although several proposals have been made, have not yet been absolutely identified. In particular, one of these domains, located within the cytoplasmic loop in the area known as the 'hinge' domain, exhibits the highest degree of conservation. In the sarcoplasmic reticulum Ca(2+)-ATPase (SERCA-1), this region is located at residues 700-712. Comparison of the sequence in this domain with calcium-binding proteins reveals similarities with the center of the helix-loop-helix EF-hand structure that is known to form divalent-cation-binding sites. A 38-residue polypeptide, corresponding to the domain 682-719 of the Ca(2+)-ATPase was synthesized and tested for its ability to bind divalent cations. Circular-dichroism, intrinsic-fluorescence and fluorescence-energy-transfer studies performed on this polypeptide in solution support the hypothesis that this domain has, in the protein, the ability to bind a divalent cation, presumably Mg2+, with an affinity of 10-15 mM. This property is observed for the isolated polypeptide in aqueous solvent and in the presence of low concentrations of the alpha-helix promoter 2,2,2-trifluoroethanol. Substitution of either one or two critical amino acids in the sequence induces a significant reduction of the binding properties. It is proposed that this sequence is involved in the co-ordination of a Mg2+ in the nucleotide-binding site and/or in the phosphorylation site of P-type ATPases.
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PMID:Localization of a putative magnesium-binding site within the cytoplasmic domain of the sarcoplasmic reticulum Ca(2+)-ATPase. 822 59

When the SERCA 2 isoform of the Ca(2+)-ATPase of cardiac and slow-twitch muscle sarcoplasmic reticulum was coexpressed with phospholamban in COS-1 cells, a reduction in Ca2+ affinity (measured as Ca2+ dependence of Ca2+ transport) of 0.2-0.3 pCa units was observed. This inhibitory effect was reversed by phosphorylation of phospholamban with cAMP-dependent protein kinase A. SERCA 1 and SERCA 3, were also expressed in COS-1 cells, alone and together with phospholamban. SERCA 1 had high Ca2+ affinity which was reduced upon coexpression with phospholamban, but SERCA 3 had lower Ca2+ affinity, which was unaltered by coexpression with phospholamban. To identify which regions of the Ca2+ ATPase sequence determine its functional interaction with phospholamban, chimeric Ca(2+)-ATPases between SERCA 2 and SERCA 3 were constructed and coexpressed with phospholamban. Measurement of Ca2+ affinities for a series of chimeras showed that two separate regions of the cytoplasmic domain of SERCA 2 were required for manifestation of a functional interaction between phospholamban and the Ca(2+)-ATPase. The first is a region between amino acids 336 and 412 in the phosphorylation domain, which corresponds to a phospholamban interaction site identified earlier (James, P., Inui, M., Tada, M., Chiesi, M., and Carafoli, E. (1989) Nature 342, 90-92). The second region is the nucleotide binding/hinge domain (amino acids 467-762) which determines high Ca2+ affinity for SERCA type pumps (Toyofuku, T., Kurzydlowski, K., Lytton, J., and MacLennan, D. H. (1992) J. Biol. Chem. 267, 14490-14496).
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PMID:Identification of regions in the Ca(2+)-ATPase of sarcoplasmic reticulum that affect functional association with phospholamban. 842 55

The muscle and species-specific differences in enzymatic activity between Placopecten and Argopecten striated and catch muscle myosins are attributable to the myosin heavy chain. To identify sequences that may modulate these differences, we cloned and sequenced the cDNA encoding the myosin heavy chains of Placopecten striated and catch muscle. Deduced protein sequences indicate two similar isoforms in catch and striated myosins (97% identical); variations arise by differential RNA splicing of five alternative exons from a single myosin heavy chain gene. The first encodes the phosphate-binding loop; the second, part of the ATP binding site; the third, part of the actin binding site; the fourth, the hinge in the rod; and the fifth, a tailpiece found only in the catch muscle myosin heavy chain. Both Placopecten myosin heavy chains are 96% identical to Argopecten myosin heavy chaina isoforms. Because subfragment-1 ATPase activities reflect the differences observed in the parent myosins, the motor domain is responsible for the variations in ATPase activities. In addition, data show that differences are due to Vmax and not actin affinity. The sequences of all four myosin heavy chain motor domains diverge only in the flexible surface loop near the nucleotide binding pocket. Thus, the different ATPase activities of four molluscan muscle myosins are likely due to myosin heavy chain sequence variations within the flexible surface loop that forms part of the ATP binding pocket of the motor domain.
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PMID:Sequence variations in the surface loop near the nucleotide binding site modulate the ATP turnover rates of molluscan myosins. 890 22

We have investigated the structural changes that occur in the molecular motor kinesin during its ATPase cycle, utilizing two bacterially expressed constructs. The structure of both constructs has been examined as a function of the nature of the nucleotide intermediate occupying the active site by means of sedimentation velocity, sedimentation equilibrium, fluorescence solute quenching, fluorescence anisotropy decay, and limited proteolysis. While the molecular weight of monomeric and dimeric human kinesin constructs, as measured by sedimentation velocity and sedimentation equilibrium, and the tryptic cleavage pattern are unaffected by the nucleotide intermediate occupying the active site, significant changes in the rotational correlation time of fluorescently labeled kinesin-nucleotide intermediates can be detected. These results suggest that kinesin contains an internal "hinge" whose flexibility varies through the course of the ATPase cycle. In prehydrolytic, "strong" binding states, this hinge is relatively rigid, while in posthydrolytic, "weak" binding states, it is more flexible. Our results, in conjunction with anisotropy decay studies of myosin, suggest that these two molecular motors may share a common structural feature; viz. weak binding states are characterized by segmental flexibility, which is lost upon assumption of a strong binding conformation.
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PMID:Structural studies of kinesin-nucleotide intermediates. 893 73

Treatment of rabbit sarcoplasmic reticulum Ca2+-ATPase with a variety of proteases, including elastase, proteinase K, and endoproteinases Asp-N and Glu-C, results in accumulation of soluble fragments starting close to the ATPase phosphorylation site Asp351 and ending in the Lys605-Arg615 region, well before the conserved sequences generally described as constituting the "hinge" region of this P-type ATPase (residues 670-760). These fragments, designated as p29/30, presumably originate from a relatively compact domain of the cytoplasmic head of the ATPase. They retain two structural characteristics of intact Ca2+-ATPase as follows: high sensitivity of peptidic bond Arg505-Ala506 to trypsin cleavage, and high reactivity of lysine residue Lys515 toward the fluorescent label fluorescein 5'-isothiocyanate. Regarding functional properties, these fragments retain the ability to bind nucleotides, although with reduced affinity compared with intact Ca2+-ATPase. The fragments also bind Nd3+ ions, leaving open the possibility that these fragments could contain the metal-binding site(s) responsible for the inhibitory effect of lanthanide ions on ATPase activity. The p29/30 soluble domain, like similar proteolytic fragments that can be obtained from other P-type ATPases, may be useful for obtaining three-dimensional structural information on the cytosolic portion of these ATPases, with or without bound nucleotides. From our findings we infer that a real hinge region with conformational flexibility is located at the C-terminal boundary of p29/30 (rather than in the conserved region of residues 670-760); we also propose that the ATP-binding cleft is mainly located within the p29/30 domain, with the phosphorylation site strategically located at the N-terminal border of this domain.
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PMID:Characterization of a protease-resistant domain of the cytosolic portion of sarcoplasmic reticulum Ca2+-ATPase. Nucleotide- and metal-binding sites. 950 58

In current topological models, the sarcoplasmic reticulum Ca2+-ATPase contains 10 putative transmembrane spans (M1-M10), with spans M4/M5/M6 and probably M8 participating in the formation of the membranous calcium-binding sites. We describe here the conformational properties of a synthetic peptide fragment (E785-N810) encompassing the sixth transmembrane span (M6) of Ca2+-ATPase. Peptide M6 includes three residues (N796, T799, and D800) out of the six membranous residues critically involved in the ATPase calcium-binding sites. 2D-NMR experiments were performed on the M6 peptide selectively labeled with 15N and solubilized in dodecylphosphocholine micelles to mimic a membrane-like environment. Under these conditions, M6 adopts a helical structure in its N-terminal part, between residues I788 and T799, while its C-terminal part (G801-N810) remains disordered. Addition of 20% trifluoroethanol stabilizes the alpha-helical N-terminal segment of the peptide, and reveals the propensity of the C-terminal segment (G801-L807) to form also a helix. This second helix is located at the interface or in the aqueous environment outside the micelles, while the N-terminal helix is buried in the hydrophobic core of the micelles. Furthermore, the two helical segments of M6 are linked by a flexible hinge region containing residues T799 and D800. These conformational features may be related to the transient formation of a Schellman motif (L797VTDGL802) encoded in the M6 sequence, which probably acts as a C-cap of the N-terminal helix and induces a bend with respect to the helix axis. We propose a model illustrating two conformations of M6 and its insertion in the membrane. The presence of a flexible region within M6 would greatly facilitate concomitant participation of all three residues (N796, T799, and D800) believed to be involved in calcium complexation.
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PMID:NMR conformational study of the sixth transmembrane segment of sarcoplasmic reticulum Ca2+-ATPase. 1023 32


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