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)

Trypsin digestion of phosphorylated and 3H-labeled dinitrophenylated chicken gizzard myosin released major fragments of Mr 29,000, 50,000 and 66,000 in a ratio of close to one to one. They contained 58% of the label bound to thiols of the heavy chains; 28% of the label was bound to the light chains. The heavy chain fragments of Mr 29,000 and Mr 66,000 were dinitrophenylated when the enzyme activity was inhibited. The 3H-labeled dinitrophenylated myosin alone followed a somewhat different pattern in that the label was bound to the light chains predominantly. Thiolysis of the phosphorylated and dinitrophenylated myosin with 2-mercaptoethanol restored the K+ -ATPase (ATP phosphohydrolase, EC 3.6.1.32) activity and the dinitrophenyl group was removed from the N-terminal fragment of Mr 29,000 of the heavy chain, predominantly. In contrast, restoration of the enzymic activity occurred in thiolyzed dinitrophenylated myosin alone when the label was removed from the light chains rather than the tryptic fragments of the heavy chain. Phosphorylation induced conformational changes in gizzard myosin that altered the reactivity of the thiols in fragments of the globular heavy chain region.
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PMID:Dinitrophenylated thiols in tryptic fragments of the heavy chain from chicken gizzard myosin. 379 Jan 40

A procedure for the purification of Mg(2+)-Ca(2+) adenosinetriphosphatase (EC 3.6.1.3) from E. coli, yielding relatively large amounts of highly active enzyme, is described. The enzyme consists of four nonidentical subunits. Trypsin treatment of purified enzyme yields a preparation consisting exclusively of the two larger subunits, which are sufficient for ATPase activity. Purified enzyme is inhibited by 7-chloro-4-nitrobenzo-2-oxa-1,3-diazole; this inhibition is reversed by dithiothreitol, and the diazole is found preferentially associated with the beta-subunit of the enzyme. Antibody prepared against the trypsin-treated enzyme inhibited various ATP-dependent reactions as well as membrane-bound ATPase itself.
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PMID:Purification and properties of Mg2+-Ca2+ adenosinetriphosphatase from Escherichia coli. 427 24

The F1F0 H+-ATPase in membranes of Escherichia coli was amplified by heat induction of a lysogenic lambda-unc+ transducing phage. Inverted membrane vesicles were stripped of the F1 sector of the ATPase complex by washing with EDTA. The stripped membranes were treated with dithiobis(succimidylpropionate) to cross-link subunits of the F0 sector of the ATPase complex. After electrophoresis under nonreducing conditions in one dimension, cross-linked subunits were identified by off-diagonal electrophoresis in a second dimension following cleavage of the cross-linked products with beta-mercaptoethanol. A psi-psi dimer was the major cross-linked product identified. In addition, a chi-psi product and chi-psi2 product were identified. These results support the proposed chi-psi2 stoichiometry of subunits in F0. When the F1-stripped membranes were treated with trypsin, the psi subunit was rapidly degraded, whereas psi was protected from degradation when F1 was bound to the membrane. Trypsin-treated, stripped membranes, lacking an intact psi subunit, did not bind the F1 portion of the ATPase with high affinity. However, these trypsin-treated stripped membranes remained as permeable to protons as untreated stripped membranes, and the H+ conductivity was blocked by dicyclohexylcarbodiimide. These results indicate that the portion of the psi subunit exposed on the cytoplasmic face of the inner membrane is involved in the binding of the F1 portion of the ATPase, but is not necessary for H+ conduction mediated by the F0 sector of the complex.
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PMID:Topology, organization, and function of the psi subunit in the F0 sector of the H+-ATPase of Escherichia coli. 622 25

Limited digestion of Acanthamoeba myosin II by trypsin selectively cleaved the 185,000-Da heavy chains into a 73,000-Da peptide containing the catalytic and actin-binding sites and a 112,000-Da peptide containing the regulatory phosphorylatable sites. The light chains were unaffected. The proteolytic products remained associated and formed bipolar filaments that were very similar in appearance to filaments of native myosin by negative staining electron microscopy. Filaments of trypsin-cleaved, dephosphorylated myosin, however, had a smaller sedimentation coefficient than filaments of native dephosphorylated myosin. Trypsin-cleaved dephosphorylated myosin retained complete Ca2+-ATPase activity but had no actin-activated ATPase activity under conditions that are optimal for native, dephosphorylated myosin (pH 7.0, 4 mM MgCl2, 30 degrees C or pH 6.4, 1 mM MgCl2, 30 degrees C). Trypsin-cleaved dephosphorylated myosin had higher actin-activated ATPase activity at pH 6.0 and 1 mM MgCl2 than undigested dephosphorylated myosin which is appreciably inhibited under these conditions. Trypsin-cleaved, dephosphorylated myosin inhibited the actin-activated ATPase activity of native, dephosphorylated myosin when both were present in the same co-polymers, when enzymatic activity was assayed at pH 7.0, 4 mM MgCl2, and 30 degrees C, but this inhibition was overcome by raising the MgCl2 to 6 mM. These results provide additional evidence that regulation of acanthamoeba myosin II occurs at the filament level and that, under most conditions of assay, the heavy chains must be intact and the regulatory serines unphosphorylated for actin-activated ATPase activity to be maximally expressed.
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PMID:Effects of limited tryptic cleavage on the physical and enzymatic properties of myosin II from Acanthamoeba castellanii. 623 25

The effects of trypsin digestion and low temperature on Ca2+ binding and on Ca2+ activation of ATP hydrolysis by the high-affinity transport sites of the Ca2+, Mg2+-ATPase of sarcoplasmic reticulum were examined. Sarcoplasmic reticulum vesicles contain 0.7-1.1 high-affinity Ca2+ sites per 10(5) g sarcoplasmic reticulum with K = 3-5 X 10(5) M-1, as well as sites of lower affinity. The first cleavage of the ATPase with trypsin (TD1) has no effect on the binding properties of the high affinity sites. The second tryptic cleavage (TD2) decreases the affinity of the high sites to K = 3 X 10(4) M-1 with conservation of the total number of sites. The purified ATPase contains 1.6-2.0 high affinity Ca2+ sites per 10(5) g protein when measured at 23 degrees C, while at 0-4 degrees C there is approximately equal to 1 high-affinity (K = 5 - 10 X 10(5) M-1) affinity site and approximately equal to 1 intermediate-affinity (K = 3 X 10(4) M-1) site per 10(5) g. Trypsin digestion to the point of TD1 has no effect on either the number or the binding constants of the high-affinity sites. Upon TD2 cleavage, one of the sites is converted to the intermediate-affinity state, while the other remains at high affinity. After TD2 modification of the enzyme both of the sites are in the intermediate affinity state at 4 degrees C. On the basis of the binding data, several models for the roles of the Ca2+ sites in the activation of ATP hydrolysis are derived. The results are summarized by a scheme in which the two high-affinity Ca2+ sites are heterogeneous with respect to sensitivity to temperature and to TD2 modification. The results of this and a previous study [Scott, T. L. and Shamoo, A. E. (1982) J. Membr. Biol. 64, 137-144] indicate that while occupation of either of the two Ca2+ sites can stimulate ATP hydrolysis, the site which is sensitive to TD2 is essential for the coupling of hydrolysis to Ca2+ transport.
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PMID:Distinction of the roles of the two high-affinity calcium sites in the functional activities of the Ca2+-ATPase of sarcoplasmic reticulum. 623 83

The sensitivity of the (Na+ + K+)-ATPase in human red cell membranes to inhibition by Ca2+ is markedly increased by the addition of diluted cytoplasm from hemolyzed human red blood cells. The concentration of Ca2+ causing 50% inhibition of the (Na+ + K+)-ATPase is shifted from greater than 50 microM free Ca2+ in the absence of hemolysate to less than 10 microM free Ca2+ when hemolysate diluted 1:60 compared to in vivo concentrations is added to the assay mixture. Boiling the hemolysate destroys its ability to increase the sensitivity of the (Na+ + K+)-ATPase to Ca2+. Proteins extracted from the membrane in the presence of EDTA and concentrated on an Amicon PM 30 membrane increased the sensitivity of the (Na+ + K+)-ATPase to Ca2+ in a dose-dependent fashion, causing over 80% inhibition of the (Na+ + K+)-ATPase at 10 microM free Ca2+ at the highest concentration of the extract tested. The active factor in this membrane extract is Ca2+-dependent, because it had no effect on the (Na+ + K+)-ATPase in the absence of Ca2+. Trypsin digestion prior to the assay destroyed the ability of this protein extract to increase the sensitivity of the (Na+ + K+)-ATPase to Ca2+.
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PMID:Effect of hemolysate on calcium inhibition of the (Na+ + K+)-ATPase of human red blood cells. 630 94

Purified Na+,K+-ATPase is treated with trypsin. The altered enzyme is then reconstituted into liposomes and the change in active and passive Na+,K+-fluxes is recorded. Trypsin treatment transforms the slow passive Na+,K+-fluxes into leaks. The leak formation is correlated with the degree of proteolysis and the associated decrease in Na+,K+-ATPase activity. The active Na+,K+-transport capacity decreases in parallel with the passive transport. It is thus proposed that the Na+,K+-ATPase molecule primarily contains unspecific transmembrane tunnels that are rendered ion-selective by transverse bars of specific length (bar model).
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PMID:A bar model for the pump and channel function of the reconstituted Na+,K+-ATPase. 630 26

Membrane fusion in vitro between Golgi apparatus- and plasma-membrane-rich fractions isolated from maize (Zea mays) roots was found to be dependent on Ca2+ and the membrane proteins. Trypsin treatment of mixed membrane fractions before the addition of Ca2+ inhibited their ability to fuse. It resulted also in a selective and progressive elimination of a characteristic intense polypeptide band (B1) on gel electrophoresis. This polypeptide was not removed by chymotrypsin or thermolysin. B1 is an integral membrane protein with an exposed portion to the outside. Sodium deoxycholate was used to solubilize the proteins of mixed membrane fractions. Extracted proteins analysed by non-SDS (sodium dodecyl sulphate) polyacrylamide-gel electrophoresis revealed the presence of four isolated bands. When re-electrophoresed in the presence of SDS, one of these bands exhibited the same mobility as polypeptide B1. Enzymic staining of non-SDS-polyacrylamide gels showed that this protein has Ca2+- and Mg2+-dependent ATPase activity. Its possible role in membrane fusion is discussed.
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PMID:The extraction from maize (Zea mays) root cells of membrane-bound protein with Ca2+-dependent ATPase activity and its possible role in membrane fusion in vitro. 645 76

The activation by proteases of the Ca2+-dependent ATPase of chloroplast coupling factor 1 (CF1) has been investigated. Using low concentrations of papain and trypsin, the increase in ATPase activity and the degradation of the five subunits of CF1 were compared. Sodium dodecyl sulfate-gel electrophoresis of protease-treated CF1 revealed that the delta subunit was very rapidly degraded and that the alpha and beta subunits were clipped. The gamma and epsilon subunits were more resistant to digestion. The modification of the alpha subunit of latent CF1 most closely correlated with the activation of Ca2+-ATPase activity. Trypsin treatment of dithiothreitol-activated CF1 resulted in a very rapid increase in Ca2+-ATPase activity and a corresponding rapid cleavage of the gamma subunit to a 25,000-dalton species. With more prolonged treatment, the 25,000-dalton species was cleaved to fragments of 14,000 and 11,000-daltons. Dithiothreitol treatment did not alter the rate of attack on the other subunits. The gamma subunit of heat-activated CF1 was also more susceptible to protease digestion. The increased protease sensitivity of the gamma subunit of soluble CF1 after treatment with dithiothreitol or heat mimics the increased protease sensitivity of the gamma subunit of bound CF1 when thylakoids are treated with trypsin during illumination (Moroney, J. V., and McCarty, R. E. (1982) J. Biol. Chem. 257, 5915-5920). These results suggest that the conformational changes that occur when purified CF1 is exposed to dithiothreitol are similar to those that CF1 bound to thylakoid membranes undergoes under illumination.
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PMID:Effect of proteolytic digestion on the Ca2+-ATPase activity and subunits of latent and thiol-activated chloroplast coupling factor 1. 646 50

The turmeric anti-oxidant protein (TAP) had been isolated from the aqueous extract of turmeric. The anti-oxidant principle was found to be a heat stable protein. Trypsin treatment abolished the anti-oxidant activity. The anti-oxidant principle had an absorbance maximum at 280 nm. After gel filtration, the protein showed a 2-fold increase in anti-oxidant activity and showed 2 bands in the SDS-PAGE with approximate molecular weight range of 24,000 Da. The protein showed a concentration-dependent inhibitory effect on the promoter induced lipid peroxidation. A 50% inhibitory activity of lipid peroxidation was observed at a protein concentration of 50 micrograms/ml. Ca(2+)-ATPase of rat brain homogenate was protected to nearly 50% of the initial activity from the lipid peroxidant induced inactivation by this protein. This protection of Ca(2+)-ATPase activity was found to be associated with the prevention of loss of -SH groups.
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PMID:The anti-oxidant activity of turmeric (Curcuma longa). 750 Jun 37


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