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)

Incubation of plasma membranes isolated from bovine aorta with either 0.5 mM CaCl2 or with a phorbol ester (1 microM phorbol 12,13-dibutyrate) and phosphatidylserine in an EGTA-containing buffer resulted in the phosphorylation of 10 proteins (Mr of 158, 105, 75, 62, 44, 39, 33, 22, 15 and 9 kDa), presumably due to activation of endogenous protein kinase C (PKC). After heat treatment of the aortic plasma membranes at 80 degrees C for 5 min in order to inactivate all endogenous protein kinase, phosphatase and ATPase activities, membrane phosphorylation was absolutely-dependent upon the addition of an exogenous, partially-purified PKC preparation from bovine aorta. Under these conditions, a total of 17 phosphoproteins could be detected (Mr of 158, 105, 75, 44, 39, 33, 30, 29, 27, 25, 22, 17.5, 16, 15, 11, 10 and 9 kDa). The most prominent phosphoprotein band in native membranes had a molecular weight of 75 kDa (p75); several characteristics suggest that p75 might be autophosphorylated PKC. The phosphorylation of aortic plasma membranes by exogenous PKC required phosphatidylserine and was calcium-dependent (10(-5) to 10(-7) M Ca2+); the addition of diolein resulted in little or no enhancement of phosphorylation. Replacement of phosphatidylserine with oleic acid resulted in the same number of phosphoproteins, but the extent of phosphorylation was diminished. The phosphorylation pattern was altered slightly if the aortic plasma membranes were isolated in the presence of 1 mM Ca2+ instead of EGTA buffers as in the standard procedure. Experiments were performed to determine if the p39 substrate of PKC in aortic plasma membranes was calpactin II (lipocortin I). Immunoblotting established that calpactin II was present in aortic plasma membranes, but there was no corresponding phosphoprotein on the autoradiographs.
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PMID:Phosphorylation of aortic plasma membranes by protein kinase C. 183 27

Human platelet myosin forms 10S and 6S conformations, and its Ca(2+)- and Mg(2+)-ATPase activities are parallel with the transition between 10S and 6S conformation, as judged by the gel filtration, intrinsic fluorescence, and viscosity methods. The 20,000-dalton myosin light chain (LC20) is phosphorylated by both myosin light chain kinase (MLC kinase) and Ca2+, phospholipid-dependent protein kinase (protein kinase C [PKC]). The phosphorylation (1 mol of phosphate/mol of LC20) by MLC kinase shifts the equilibrium toward the 6S conformation, but that by PKC does not. The prephosphorylation of myosin by PKC prevents the effect of phosphorylation by MLC kinase on actin-activated Mg(2+)-ATPase activity, but not the effect on conformational change. Inhibition of actin-activated ATPase activity by PKC is due to a decreased affinity of myosin for actin, and no change in Vmax is observed. These results suggest that sequential phosphorylation of myosin by both kinases plays an important role in the ATPase activities of human platelet myosin.
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PMID:Effect of phosphorylation of myosin light chain by myosin light chain kinase and protein kinase C on conformational change and ATPase activities of human platelet myosin. 183 91

Thapsigargin is found to be a potent inhibitor of the intracellular Ca2+ pump proteins from skeletal muscle sarcoplasmic reticulum (SR), cardiac SR, and brain microsomes. For skeletal muscle SR, the molar ratio of thapsigargin to Ca2+ pump protein for complete inhibition (MRc) of the Ca2+ loading rate, Ca(2+)-dependent ATPase activity, and formation of phosphorylated intermediate (EP) was approximately 1. When the Ca2+ pump protein of low affinity to Ca2+ (E2 state) was pretreated with thapsigargin, ATP and Ca2+ binding to the Ca2+ pump protein was completely inhibited. In the presence of Ca2+ (E1 state), Ca2+ pump protein was protected from inactivation by thapsigargin with respect to Ca2+ binding and EP formation. The MRc for brain microsomes, which mediate Ca2+ uptake into intracellular (inositol 1,4,5-trisphosphate-releasable) Ca2+ pools, is likewise stoichiometric. Approximately 30% of Ca2+ loading activity of brain microsomes was insensitive to thapsigargin, indicating the presence of other Ca2+ pumping system(s). The MRc for heart is 3.8, indicating that the Ca2+ pump of cardiac SR is less sensitive to thapsigargin. Phosphorylation of cardiac SR with protein kinase A increased the sensitivity to thapsigargin to MRc of 2.8. In summary, we find that: 1) thapsigargin is the most effective inhibitor of the Ca2+ pump protein of intracellular membranes (SR and endoplasmic reticulum); 2) its primary inhibitory action appears to inactivate the E2 form of the enzyme preferentially; 3) cardiac SR shows lesser sensitivity to thapsigargin than skeletal muscle SR and brain microsomes; protein kinase A treatment of cardiac SR enhances the sensitivity to the drug.
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PMID:Drug action of thapsigargin on the Ca2+ pump protein of sarcoplasmic reticulum. 183 73

The role of cGMP-dependent protein kinase in the regulation of intracellular Ca2+ levels in vascular smooth muscle cells was examined by studying the effects of cGMP on the phosphorylation of the Ca(2+)-ATPase regulatory protein phospholamban. Cultured rat aortic smooth muscle cells incubated with atrial natriuretic peptide II or sodium nitroprusside responded with increased phosphorylation of the 6000-Da subunit of phospholamban. The identity of phospholamban was confirmed using immunoprecipitation methods. Phosphorylation was associated with an increase in the activation of membrane-associated ATPase by Ca2+. These results indicated that at least one site of action of cGMP in smooth muscle cells is the sarcoplasmic reticulum, where phosphorylation of proteins regulating Ca2+ fluxes occurs. Studies using confocal laser scanning microscopy to define the cellular distribution of cGMP-dependent protein kinase suggested that the enzyme was localized to the same cellular region(s) as was phospholamban. Phosphorylation of proteins by cGMP in broken cell fractions from rabbit aorta was also performed. Phospholamban and other proteins were phosphorylated in the presence of cGMP but not cAMP, suggesting that only cGMP-dependent protein kinase was associated with smooth muscle membrane fractions containing phospholamban. These results suggest that one mechanism of action of cGMP in the reduction of intracellular Ca2+ is the activation of sarcoplasmic reticulum Ca(2+)-ATPase via phosphorylation of phospholamban. The data also support the concept that compartmentalization of protein kinases with substrates in the intact cell is an important factor involved in protein phosphorylation.
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PMID:Regulation of sarcoplasmic reticulum protein phosphorylation by localized cyclic GMP-dependent protein kinase in vascular smooth muscle cells. 183 34

Cyclic GMP (cGMP) mediates the relaxing action of a variety of vasodilator drugs and endogenous vasodilator substances. Cyclic AMP (cAMP) mediates relaxation by beta-adrenergic agonists as well as other activators of adenylate cyclase. Both second messengers appear to reduce the concentration of intracellular Ca2+ in vascular smooth muscle cells, thus affecting relaxation. The presence of cGMP-dependent protein kinase in vascular smooth muscle cells is required for the reduction of Ca2+ by cAMP and cGMP, suggesting that this enzyme mediates the relaxing effects of both cyclic nucleotides. Although the specific substrate proteins for cGMP-dependent protein kinase are not well characterized in vascular smooth muscle, new evidence indicates that Ca2(+)-ATPase activation by phosphorylation of phospholamban by the kinase may underlie the mechanism of action of cyclic-nucleotide-dependent relaxation.
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PMID:Towards an understanding of the mechanism of action of cyclic AMP and cyclic GMP in smooth muscle relaxation. 184 22

We have previously demonstrated [Rihs, H.-P. and Peters, R. (1989) EMBO J., 8, 1479-1484] that the nuclear transport of recombinant proteins in which short fragments of the SV40 T-antigen are fused to the amino terminus of Escherichia coli beta-galactosidase is dependent on both the nuclear localization sequence (NLS, T-antigen residues 126-132) and a phosphorylation-site-containing sequence (T-antigen residues 111-125). While the NLS determines the specificity, the rate of transport is controlled by the phosphorylation-site-containing sequence. The present study furthers this observation and examines the role of the various phosphorylation sites. Purified, fluorescently labeled recombinant proteins were injected into the cytoplasm of Vero or hepatoma (HTC) cells and the kinetics of nuclear transport measured by laser microfluorimetry. By replacing serine and threonine residues known to be phosphorylated in vivo, we identified the casein kinase II (CK-II) site S111/S112 to be the determining factor in the enhancement of the transport. Either of the residues 111 or 112 was sufficient to elicit the maximum transport enhancement. The other phosphorylation sites (S120, S123, T124) had no influence on the transport rate. Examination of the literature suggested that many proteins harboring a nuclear localization sequence also contain putative CK-II sites at a distance of approximately 10-30 amino acid residues from the NLS. CK-II has been previously implicated in the transmission of growth signals to the nucleus. Our results suggest that CK-II may exert this role by controlling the rate of nuclear protein transport.
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PMID:The rate of nuclear cytoplasmic protein transport is determined by the casein kinase II site flanking the nuclear localization sequence of the SV40 T-antigen. 184 77

Advances in regulation by secondary messengers of Ca2+ level in cardiomyocyte and vascular smooth muscle cell cytosols with special reference to the major differences in regulatory effects in cells of the both types are reviewed. The effects of cAMP, cGMP, Ca2+, calmodulin, diacylglycerol and polyphosphoinositides on the Ca(2+)-channel, Ca(2+)-ATPase, plasmalemma, sarcoplasmic reticulum and outer membrane Na+/Ca2+ uniporter function are considered. Compartmentation of secondary messengers and protein kinase in cardiac and vascular smooth muscle cells should be taken into consideration during extrapolation of in vitro data to an in situ situation. The feasible role of impaired phosphorylation of membrane-bound proteins of cardiac and vascular smooth muscle cells in cardiac insufficiency and atherosclerosis is discussed.
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PMID:[Second messengers in heart cells and smooth muscle vessels]. 191 66

Monoclonal and polyclonal antibodies to the major sarcoplasmic reticulum proteins of rabbit skeletal and canine cardiac muscle have been used to identify and characterize the corresponding components of human cardiac sarcoplasmic reticulum. The Ca2(+)-transporting ATPase of human cardiac sarcoplasmic reticulum was identified as a 105,000-Da protein antigenically distinct from its rabbit skeletal muscle counterpart. Human cardiac sarcoplasmic reticulum also contained 53,000- 155,000- and 165,000-Da glycoproteins antigenically related to the low and high molecular weight glycoproteins of canine cardiac and rabbit skeletal muscle sarcoplasmic reticulum. The ryanodine-sensitive Ca2+ channel of human cardiac sarcoplasmic reticulum was identified as a 400,000-Da protein antigenically related to its counterparts in canine cardiac and rabbit skeletal muscle. Human cardiac calsequestrin was identified as a 52,000-Da protein. Human phospholamban was identified as a 29,000-Da substrate for phosphorylation by cAMP-dependent protein kinase. Immunoblots of sarcoplasmic reticulum from the normal left ventricles of four unmatched organ donors and the excised failing left ventricles of nine patients with idiopathic dilated cardiomyopathy were compared in search of qualitative differences in the protein patterns of the failing hearts. No such differences were found with respect to the Ca2+ ATPase, the 53,000-Da glycoprotein, the ryanodine-sensitive Ca2+ channel, calsequestrin or phospholamban. In contrast, the 165,000-Da glycoprotein band, present in all four preparations from nonfailing hearts, was absent from three of nine preparations from failing hearts, and staining of the 155,000-Da glycoprotein in these three preparations appeared to be relatively increased. The absence of the 165,000-Da glycoprotein band may identify or reflect a pathogenetic mechanism in a subset of patients with idiopathic dilated cardiomyopathy.
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PMID:Identification and characterization of proteins in sarcoplasmic reticulum from normal and failing human left ventricles. 208 60

The Ca2(+)-ATPase in cardiac sarcoplasmic reticulum (SR) is under regulation by phospholamban, an oligomeric proteolipid. To determine the molecular mechanism by which phospholamban regulates the Ca2(+)-ATPase, a reconstitution system was developed, using a freeze-thaw sonication procedure. The best rates of Ca2+ uptake (700 nmol/min/mg reconstituted vesicles compared with 800 nmol/min/mg SR vesicles) were observed when cholate and phosphatidylcholine were used at a ratio of cholate/phosphatidylcholine/Ca2(+)-ATPase of 2:80:1. The EC50 values for Ca2+ were 0.05 microM for both Ca2+ uptake and Ca2(+)-ATPase activity in the reconstituted vesicles compared with 0.63 microM Ca2+ in native SR vesicles. Inclusion of phospholamban in the reconstituted vesicles was associated with a significant inhibition of the initial rates of Ca2+ uptake at pCa 6.0. However, phosphorylation of phospholamban by the catalytic subunit of the cAMP-dependent protein kinase reversed the inhibitory effect on the Ca2+ pump. Similar findings were observed when a peptide, corresponding to amino acids 1-25 of phospholamban, was used. These findings indicate that phospholamban is an inhibitor of the Ca2(+)-ATPase in cardiac SR and phosphorylation of phospholamban relieves this inhibition. The mechanism by which phospholamban inhibits the Ca2+ pump is unknown, but our findings with the synthetic peptide suggest that a direct interaction between the Ca2(+)-ATPase and the hydrophilic portion of phospholamban may be one of the mechanisms for regulation.
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PMID:Functional reconstitution of the cardiac sarcoplasmic reticulum Ca2(+)-ATPase with phospholamban in phospholipid vesicles. 213 56

The sequence of more than 1,000 amino acid residues, derived from two different isoforms, has been determined from peptides generated from purified human erythrocyte membrane Ca2(+)-ATPase (hPMCA). Several of these peptide sequences correspond to the previously reported, cDNA deduced sequence of the "teratoma" Ca2+ pump isoform hPMCA1 (Verma, A. K., Filoteo, A. G., Stanford, D. R., Wieben, E. D., Penniston, J. T., Strehler, E. E., Fischer, R., Heim, R., Vogel, G., Matthews, S., Strehler-Page, M.-A., James, P., Vorherr, T., Krebs, J., and Carafoli, E. (1988) J. Biol. Chem. 263, 14152-14159). The complete primary structure of a novel isoform (hPMCA3) has been determined by molecular cloning and nucleotide sequencing of its corresponding cDNA. This new member of the plasma membrane Ca2+ pump family consists of 1,205 amino acid residues with a calculated Mr of 133,930, and it shows 88% similarity (75% identity) with the previously sequenced pump isoform. Specific probes detect major mRNA species of 5.6 kilobases for hPMCA1, and of 7.5 kilobases for hPMCA3, on Northern blots of human K562 erythroleukemic cell RNA. A large number of peptide sequences match perfectly with only one or the other of these isoforms and all peptides (with 6 exceptions corresponding to a contaminant protein or to a third minor Ca2+ pump isoform) are found in either only one or in both of the isoforms. The two erythrocyte Ca2+ pumps display high sequence divergence in a few localized regions that may determine isoform-specific functional specializations; for example, the putative extracellular loop separating transmembrane domains 1 and 2, the highly negatively charged region previously suggested to be involved in Ca2+ binding, and the site of cAMP-dependent protein kinase phosphorylation.
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PMID:Peptide sequence analysis and molecular cloning reveal two calcium pump isoforms in the human erythrocyte membrane. 213 51


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