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)

The Ca(2+)-ATPase of skeletal sarcoplasmic reticulum was purified and reconstituted in proteoliposomes containing phosphatidylcholine (PC). When reconstitution occurred in the presence of PC and the acidic phospholipids, phosphatidylserine (PS) or phosphatidylinositol phosphate (PIP), the Ca(2+)-uptake and Ca(2+)-ATPase activities were significantly increased (2-3 fold). The highest activation was obtained at a 50:50 molar ratio of PS:PC and at a 10:90 molar ratio of PIP:PC. The skeletal SR Ca(2+)-ATPase, reconstituted into either PC or PC:PS proteoliposomes, was also found to be regulated by exogenous phospholamban (PLB), which is a regulatory protein specific for cardiac, slow-twitch skeletal, and smooth muscles. Inclusion of PLB into the proteoliposomes was associated with significant inhibition of the initial rates of Ca(2+)-uptake, while phosphorylation of PLB by the catalytic subunit of cAMP-dependent protein kinase reversed the inhibitory effects. The effects of PLB on the reconstituted Ca(2+)-ATPase were similar in either PC or PC:PS proteoliposomes, indicating that inclusion of negatively charged phospholipid may not affect the interaction of PLB with the skeletal SR Ca(2+)-ATPase. Regulation of the Ca(2+)-ATPase appeared to involve binding with the hydrophilic portion of phospholamban, as evidenced by crosslinking experiments, using a synthetic peptide which corresponded to amino acids 1-25 of phospholamban. These findings suggest that the fast-twitch isoform of the SR Ca(2+)-ATPase may be also regulated by phospholamban although this regulator is not expressed in fast-twitch skeletal muscles.
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PMID:Regulation of the skeletal sarcoplasmic reticulum Ca(2+)-ATPase by phospholamban and negatively charged phospholipids in reconstituted phospholipid vesicles. 146 Dec 59

We studied changes in myofibrillar function and protein profiles after complete global ischemia with anoxia in rat hearts. Hearts were exposed to global ischemia and anoxia (CGI) for 30 or 60 minutes at 37 degrees C, and myofibrils were prepared for measurement of Ca(2+)-dependent Mg(2+)-ATPase activity at pH 7.0 and 6.5. Hearts incubated in cold saline (1 +/- 1 degrees C) and nonincubated hearts served as controls. Maximum ATPase activity was unchanged at pH 7.0 and pH 6.5 in myofibrils from hearts treated with 30 or 60 minutes of CGI. At pH 7.0, the Hill coefficient, which is an index of cooperative interactions among thin-filament proteins, was unchanged after 30 minutes of CGI but was significantly increased after 60 minutes of CGI. A similar trend for increased cooperativity was observed when myofibrillar ATPase activity was measured at pH 6.5 in myofibrils from rat hearts made ischemic for 30 or 60 minutes. Both 30 and 60 minutes of CGI resulted in increased pCa50 values (half-maximally activating free [Ca2+]) at pH 7.0 and pH 6.5. Densitometric analysis of myofibrillar proteins separated with sodium dodecyl sulfate-polyacrylamide gel electrophoresis indicated that troponin I and troponin T were degraded during 60 minutes of CGI. Two new protein bands appearing in ischemia-treated myofibrils were identified as partially degraded troponin I and troponin T with Western blots. The troponin I fragment could be phosphorylated by cAMP-dependent protein kinase. In addition, we observed phosphorylation of a protein band that corresponded to myosin light chain-2 in myofibrils from CGI-treated hearts. These results suggest that degradation of thin-filament proteins may contribute to the changes in cooperativity of Ca2+ regulation of ATPase activity observed in the myofibrils from rat hearts exposed to CGI.
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PMID:Alterations in myofibrillar function and protein profiles after complete global ischemia in rat hearts. 153 Nov 86

A nucleoside triphosphatase (NTPase) activity appeared to be associated with a highly purified nuclear preparation from rat cardiac ventricles. Different nucleoside triphosphates (UTP greater than GTP greater than ITP greater than CTP) supported this enzymic activity, which was stimulated by Mg2+ but not by Ca2+. The nuclear NTPase activity could be down regulated by endogenous phosphorylation of a 55,000 Mr protein. Maximal phosphorylation of the 55,000 Mr protein occurred in the presence of Mg(2+)-ATP. Addition of cAMP, cGMP, Ca2+, Ca2+/phospholipid, Ca2+/calmodulin, and catalytic subunit of cAMP-dependent protein kinase was not associated with any further phosphorylation of the 55,000 Mr protein. However, in the presence of Ca2+/calmodulin or the catalytic subunit of the cAMP-dependent protein kinase additional proteins became phosphorylated, but these had no effect on the Mg(2+)-NTPase activity. These results indicate that a protein with Mr 55,000 may be involved in the regulation the Mg(2+)-NTPase activity associated with rat cardiac nuclei.
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PMID:Regulation of rat cardiac nuclei-associated Mg(2+)-NTPase by phosphorylation. 165 81

We have examined two distinct protein kinases, cAMP-dependent protein kinase and protein kinase C, for their ability to phosphorylate and regulate the activity of three different types of Na+,K(+)-ATPase preparation. cAMP-dependent protein kinase phosphorylated purified shark rectal gland Na+,K(+)-ATPase to a stoichiometry of approximately 1 mol of phosphate per mol of alpha subunit. Protein kinase C phosphorylated purified shark rectal gland Na+,K(+)-ATPase to a stoichiometry of approximately 2 mol of phosphate per mol of alpha subunit. The phosphorylation by each of the kinases was associated with an inhibition of Na+,K(+)-ATPase activity of about 40-50%. These two protein kinases also inhibited the activity of a partially purified preparation of Na+,K(+)-ATPase from rat renal cortex and the activity of Na+,K(+)-ATPase present in preparations of basolateral membrane vesicles from rat renal cortex.
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PMID:Phosphorylation of the catalytic subunit of Na+,K(+)-ATPase inhibits the activity of the enzyme. 166 94

A monoclonal antibody against phospholamban has been reported to increase Ca2+ uptake by cardiac sarcoplasmic reticulum. We compared the effect of this antibody on Ca2+ pump ATPase activity of cardiac sarcoplasmic reticulum vesicles to the effect of cAMP-dependent phosphorylation of phospholamban. The antibody markedly stimulated the Ca(2+)-dependent ATPase activity in parallel to the increase in Ca2+ uptake by cardiac sarcoplasmic reticulum. When the Ca(2+)-dependent profile of the ATPase activity was compared, the KCa was shifted from 1.24 to 0.62 microM by the antibody, whereas cAMP-dependent phosphorylation of phospholamban shifted the KCa to 0.84 microM. When cardiac sarcoplasmic reticulum vesicles were treated with both cAMP-dependent protein kinase and the antibody, the stimulation was the same as that with the antibody alone. Thus, the Ca2+ pump ATPase seems to be fully activated by the antibody. The stoichiometry between Ca2+ uptake and ATPase rate was around 1 and no significant change was observed by the treatment with the antibody. Therefore, the stimulation of Ca2+ uptake of cardiac sarcoplasmic reticulum by the antibody occurred by the stimulation of Ca2+ pump ATPase, not by other mechanisms such as channel activity of phospholamban. These results indicate that the binding of the antibody to phospholamban produces essentially the same mode of action on Ca2+ pump ATPase as that of phospholamban phosphorylation. The antibody and phospholamban phosphorylation appear to release the inhibitory action of phospholamban on Ca2+ pump ATPase, resulting in the stimulation of Ca2+ pump.
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PMID:Effects of monoclonal antibody against phospholamban on calcium pump ATPase of cardiac sarcoplasmic reticulum. 166 13

A protein of apparent Mr = 15,000 on sodium dodecyl sulfate-polyacrylamide gel electrophoresis is the major plasma membrane substrate for cAMP-dependent protein kinase (PK-A) and protein kinase C (PK-C) in several different tissues. In the work described here, we purified, cloned, and sequenced the canine cardiac sarcolemmal "15-kDa protein." The amino terminus of the purified protein was not blocked, allowing determination of 50 consecutive residues by standard Edman degradation. Overlapping proteolytic phosphopeptides yielded 22 additional residues at the carboxyl terminus. Dideoxy sequencing of the full-length cDNA confirmed that the 15-kDa protein contains 72 amino acids, plus a 20-residue signal sequence. The mature protein has a calculated Mr = 8409. There is one hydrophobic membrane-spanning segment composed of residues 18-37. The acidic amino-terminal end (residues 1-17) of the protein is oriented extracellularly, whereas the basic carboxyl-terminal end (residues 38-72) projects into the cytoplasm. The positively charged carboxyl terminus contains the phosphorylation sites for PK-A and PK-C. In the transmembrane region, the 15-kDa protein exhibits 52% amino acid identity with the "gamma" subunit of Na,K-ATPase. High stringency Northern blot analysis revealed that 15-kDa mRNA is present in heart, skeletal muscle, smooth muscle, and liver but absent from brain and kidney. We propose the name "phospholemman" for the 15-kDa protein, which denotes the protein's location within the plasma membrane and its characteristic multisite phosphorylation.
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PMID:Purification and complete sequence determination of the major plasma membrane substrate for cAMP-dependent protein kinase and protein kinase C in myocardium. 171 Feb 17

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

Studies in animal models have suggested that alterations affecting phospholamban-mediated stimulation of Ca2+ uptake by sarcoplasmic reticulum are involved in the pathophysiology of heart disease. A monoclonal antibody that binds to phospholamban and stimulates Ca2+ uptake was used to characterize phospholamban-mediated effects in human cardiac sarcoplasmic reticulum and to compare these effects in tissue from normal and failing hearts. Stimulation of Ca2+ uptake by anti-phospholamban monoclonal antibody simulated the effect of phosphorylation of phospholamban by cAMP-dependent protein kinase. Binding of anti-phospholamban antibody reduced the K0.5 of the Ca2(+)-transporting ATPase from 0.53 microM [( Ca2+]) to 0.29 microM [( Ca2+]), without affecting Vmax or nHill. At 0.2 microM Ca2+, stimulation was 1.93-fold in sarcoplasmic reticulum prepared from normal human left ventricular myocardium and 1.94-fold in sarcoplasmic reticulum prepared from the left ventricular myocardium of patients with heart failure resulting from idiopathic dilated cardiomyopathy. Stimulation of Ca2+ uptake in canine cardiac sarcoplasmic reticulum under identical conditions was 1.89-fold. Phospholamban-mediated stimulation of Ca2+ uptake in human cardiac sarcoplasmic reticulum is thus comparable in magnitude to that observed in other species and results from an increase in the apparent affinity of the Ca2(+)-transporting ATPase for Ca2+. The pathogenesis of heart failure in idiopathic dilated cardiomyopathy does not, however, appear to involve intrinsic alterations of this mechanism.
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PMID:Phospholamban-mediated stimulation of Ca2+ uptake in sarcoplasmic reticulum from normal and failing hearts. 213 70


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