Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: EC:3.6.3.1 (Mg2+-ATPase)
1,484 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Cardiac sarcoplasmic reticulum plays a critical role in the excitation-contraction cycle and hormonal regulation of heart cells. Catecholamines exert their ionotropic action through the regulation of calcium transport into the sarcoplasmic reticulum. Cyclic 3'-5'-adenosine monophosphate (cAMP) causes the cAMP-dependent protein kinase to phosphorylate the regulatory protein phospholamban, which results in the stimulation of calcium transport. Calmodulin also phosphorylates phospholamban by a calcium-dependent mechanism. We have reported the isolation and purification of phospholamban with low deoxycholate (DOC) concentrations (5 X 10(-6) M). We have also reported the isolation and purification of Ca2+ + Mg2+-ATPase with a similar procedure. Both phospholamban and Ca2+ + Mg2+-ATPase retained their native properties associated with sarcoplasmic reticulum vesicles. Further, we have shown that the removal of phospholamban from membranes of sarcoplasmic reticulum vesicles uncouples Ca2+-uptake from ATPase without any effect on Ca2+ + Mg2+-ATPase activity or Ca2+ efflux. Phospholamban appears to be the substrate for both the Ca2+-calmodulin system and the cAMP-dependent protein kinase system. It is found that the phosphorylation of phospholamban by the Ca2+-calmodulin system is required for the normal basal level of Ca2+ transport, and that the phosphorylation of phospholamban at another site by the cAMP-dependent protein kinase system causes the stimulation of Ca2+-transport above the basal level. The functional effects of the phosphorylation of phospholamban by cAMP-dependent protein kinase system are expressed only after the phosphorylation of phospholamban with Ca2+-calmodulin system. We propose a model for the cardiac Ca2+ + Mg2+-ATPase, whereby the enzyme is normally uncoupled from Ca2+ uptake. The enzyme becomes coupled to Ca2+ transport after the first site of phospholamban is phosphorylated with the Ca2+-calmodulin system. When the second site of phospholamban is phosphorylated with cAMP-dependent protein kinase both Ca2+ transport and ATPase are stimulated and phospholamban becomes inaccessible to DOC solubilization and trypsin.
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PMID:Role of phospholamban in regulating cardiac sarcoplasmic reticulum calcium pump. 614 39

Ca2+-Mg2+-ATPase of sarcoplasmic reticulum was subjected to trypic digestion under various conditions and the cleavage patterns were compared. The first tryptic cleavage to yield the NH2-terminal A-fragment (Mr approximately equal to 55,000) and COOH-terminal B-fragment (Mr approximately equal to 45,000) [Thorley-Lawson, D.A. & Green, N.M. (1977) Biochem. J. 167, 739-748] was little affected by adding ligands such as Ca2+ and AMP-P(NH)P. On the other hand, subsequent splitting of A-fragment into A1 (Mr approximately equal to 30,000) and A2 (Mr approximately equal to 20,000), and further cleavages giving rise to three smaller fragments of Mr approximately equal to 27,000-28,000 (A1a, A1b, and C) [Saito, K., et al. (1984) J. Biochem. 95, 1297] were profoundly affected by these ligands. A difference in cleavage sites was noted depending on Ca2+ ion concentration; thus, A1b and C were the major components remaining after digestion in the presence and absence of Ca2+, respectively. AMP-P(NH)P markedly stabilized both A1 and A2 fragments, but the effect was much more prominent when Ca2+ was simultaneously present on the transport site. These findings suggest that conformational changes of the ATPase molecule upon binding of Ca2+, AMP-P(NH)P, or both are accompanied by corresponding changes in the susceptibility to tryptic digestion. Fragments A1 and A2 were both quite stable and fragmentation did not proceed beyond A1, when sarcoplasmic reticulum membranes were treated with trypsin at 0 degrees C. Significant further fragmentation of A1 was observed only above 20 degrees C, suggesting a conformational transition of the ATPase protein around that temperature.
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PMID:Conformational change of Ca2+,Mg2+-adenosine triphosphatase of sarcoplasmic reticulum upon binding of Ca2+ and adenyl-5'-yl-imidodiphosphate as detected by trypsin sensitivity analysis. 614 6

Using slices of mouse or rat cerebral cortex incubated with [3H]adenosine or [3H]adenine and/or [14C]GABA we have examined factors affecting the release of these compounds, and especially the influence of methylxanthines. Although release of purines and GABA could be induced by ouabain (10(-4) M), or p-hydroxymercuribenzoate (5 x 10(-4) M) no release was produced by ethacrynic acid (10(-3) or 10(-4) M) phenytoin (10(-3) M), noradrenaline or SC 13504. Release is probably not therefore related to (Na+, K+) ATPase or Mg2+-ATPase inhibition. At concentrations of 10(-3) and 10(-4) M, caffeine, theophylline, aminophylline and isobutyl-methylxanthine (IBMX) markedly depressed the release of purines evoked by ouabain. Non-xanthine inhibition of phosphodiesterase had much weaker though statistically significant effects. The methylxanthines had no significant effect on GABA release. It is suggested that the results can be explained on the basis of a positive feedback system in which released adenosine activates membranal adenylate cyclase, and the increased concentration of cyclic AMP which results form or origin of much of the adenosine released subsequently. However, we cannot exclude the existence of an intracellular receptor for methylxanthines which causes directly the inhibition of purine release.
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PMID:Methylxanthines modulate adenosine release from slices of cerebral cortex. 616 26

The mechanism of transport of basic amino acids into vacuoles of cells of the yeast Saccharomyces cerevisiae was investigated in vitro. Right-side-out vacuolar membrane vesicles were prepared from purified vacuoles. Arginine was taken up effectively by the vesicles only in the presence of ATP, not in the presence of ADP or AMP-adenosyl-5'-yl imidodiphosphate. It was exchangeable and was released completely by a protonophore, 3,5-di-tert-butyl-4-hydroxybenzilidenemalononitrile (SF6847). The transport required Mg2+ ion but was inhibited by Cu2+, Ca2+, or Zn2+ ions. The transport activity was sensitive to the ATPase inhibitor N,N'-dicyclohexylcarbodiimide (DCCD), but not to oligomycin or sodium vanadate. SF6847 or nigericin blocked arginine uptake completely, but valinomycin had no effect. ATP-dependent formation of a delta pH across the membrane vesicles was shown by quenching of 9-aminoacridine fluorescence. These results indicate that DCCD-sensitive, Mg2+-ATPase of vacuolar membranes is essential as an energy-donating system for the active transport, and that an electrochemical potential difference of protons is a driving force of this basic amino acid transport. Arginine transport showed saturation kinetics with a Km value of 0.6 mM and the mechanism was well explained by an H+/arginine antiport.
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PMID:Active transport of basic amino acids driven by a proton motive force in vacuolar membrane vesicles of Saccharomyces cerevisiae. 645 Jul 64

Selective chemical modification was used to examine amino acid residues that might be critical for the operation of the gastric K+-stimulated ATPase. Modification of amino groups with the fluorigenic reagent 2-methoxy-2,4-diphenyl-3-dihydrofuranone resulted in selective inhibition of the K+-stimulated ATPase and H+-transporting activities of the gastric microsomes, while the Mg2+-atpase was not affected. Half-maximal inhibition occurred at about 3 microgram 2-methoxy-2,4-diphenyl-3-dihydrofuranone/ml at pH 8.5. ATP provided complete protection against inhibition; the apparent Km for ATP protection was about 50 microM. Nucleotide selectivity for protection was ATP greater than ADP greater than ITP greater than GTP greater than CTP greater than AMP. Sodium dodecyl sulfate gel electrophoresis of the reacted microsomes showed that virtually all the fluorescent label was on the Mr 100 000 peptide band, a very small peptide, and aminolipids. In the presence of ATP there was about 75% reduction in the fluorescent label on the Mr 100 000 peptide, but no change in the labeling of the other components. The arginine specific reagent, butanedione, inhibited Mg2+-ATPase and K+-ATPase activities, with the former being much less reactive. Similar to 2-methoxy-2,4-diphenyl-3-dihydrofuranone, ATP provided complete protection from butanedione treatment. It is concluded that amino and guanidino groups are critical to the function of the K+-ATPase and may be actually at the ATP binding site.
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PMID:Chemical modification of gastric microsomal potassium-stimulated ATPase. 738 25

We studied the ATP dependence of NHE-1, the ubiquitous isoform of the Na+/H+ antiporter, using the whole-cell configuration of the patch-clamp technique to apply nucleotides intracellularly while measuring cytosolic pH (pHi) by microfluorimetry. Na+/H+ exchange activity was measured as the Na(+)-driven pHi recovery from an acid load, which was imposed via the patch pipette. In Chinese hamster ovary (CHO) fibroblasts stably transfected with NHE-1, omission of ATP from the pipette solution inhibited Na+/H+ exchange. Conversely, ATP perfusion restored exchange activity in cells that had been metabolically depleted by 2-deoxy-D-glucose and oligomycin. In cells dialyzed in the presence of ATP, no "run-down" was observed even after extended periods, suggesting that the nucleotide is the only diffusible factor required for optimal NHE-1 activity. Half-maximal activation of the antiporter was obtained at approximately 5 mM Mg-ATP. Submillimolar concentrations failed to sustain Na+/H+ exchange even when an ATP regenerating system was included in the pipette solution. High ATP concentrations are also known to be required for the optimal function of other cation exchangers. In the case of the Na/Ca2+ exchanger, this requirement has been attributed to an aminophospholipid translocase, or "flippase.". The involvement of this enzyme in Na+/H+ exchange was examined using fluorescent phosphatidylserine, which is actively translocated by the flippase. ATP depletion decreased the transmembrane uptake of NBD-labeled phosphatidylserine (NBD-PS), indicating that the flippase was inhibited. Diamide, an agent reported to block the flippase, was as potent as ATP depletion in reducing NBD-PS uptake. However, diamide had no effect on Na+/H+ exchange, implying that the effect of ATP is not mediated by changes in lipid distribution across the plasma membrane. K-ATP and ATP gamma S were as efficient as Mg-ATP in sustaining NHE-1 activity, while AMP-PNP and AMP-PCP only partially substituted for ATP. In contrast, GTP gamma S was ineffective. We conclude that ATP is the only soluble factor necessary for optimal activity of the NHE-1 isoform of the antiporter. Mg2+ does not appear to be essential for the stimulatory effect of ATP. We propose that two mechanisms mediate the activation of the antiporter by ATP: one requires hydrolysis and is likely an energy-dependent event. The second process does not involve hydrolysis of the gamma-phosphate, excluding mediation by protein or lipid kinases. We suggest that this effect is due to binding of ATP to an as yet unidentified, nondiffusible effector that activates the antiporter.
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PMID:ATP dependence of Na+/H+ exchange. Nucleotide specificity and assessment of the role of phospholipids. 904 42

The chicken T-tubule Mg2+-ATPase is an integral membrane glycoprotein that presents properties different from those of other ATPases located in skeletal muscle cells and exhibits ATP-hydrolysing activity on the extracellular side of the transverse tubule (TT) membranes. In this study we demonstrate that TT vesicles purified from chicken skeletal muscle possess ecto-ADPase and ecto-5'-nucleotidase activities that, along with ecto-ATPase, are able to sequentially degrade extracellular ATP to ADP, AMP and adenosine. Characterization studies of these TT ectonucleotidases revealed remarkable differences between ecto-ATPase and ecto-ADPase activities with respect to thermal stability, temperature dependence of the hydrolytic activity, effect of ionic strength, kinetic behaviour, divalent cation preference and responses to azide, N-ethylmaleimide, NaSCN, Triton X-100 and concanavalin A. Ecto-ATPase, but not ecto-ADPase, was inhibited by a polyclonal antibody against the chicken TT ecto-ATPase. On the basis of these results we propose that ATP and ADP hydrolysis are accomplished by two distinct enzymes and therefore the TT ecto-ATPase is not an apyrase. 5'-Nucleotidase activity was inhibited by adenosine 5'-[alpha,beta-methylene]diphosphate and concanavalin A, followed simple Michaelis-Menten kinetics and was released from the membranes by treatment with phosphatidylinositol-specific phospholipase C, indicating that AMP hydrolysis in T-tubules is catalysed by a typical ecto-5'-nucleotidase. Results obtained from electrophoresis experiments under native conditions suggest that ecto-ATPase, ecto-ADPase and 5'-nucleotidase might be associated, forming functional complexes in the T-tubule membranes. The TT ectonucleotidases constitute an enzymic cascade for the degradation of extracellular ATP that might be involved in the regulation of purinergic signalling in the muscle fibre.
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PMID:T-tubule membranes from chicken skeletal muscle possess an enzymic cascade for degradation of extracellular ATP. 958 72

Because epidermal growth factor (EGF) is likely to be released in the glomeruli during glomerular injury and mesangial cells possess specific receptors for EGF, we thought it to be of interest to examine whether this growth factor could influence the expression of ectoenzymes in cultured human mesangial cells. EGF stimulated 5'-nucleotidase and aminopeptidase N activities in intact human mesangial cells in a time- (24-72 h) and dose-dependent (0.1-50 ng ml(-1)) manner. Maximum stimulation represented 2.7- and 2-fold basal activities for 5'-nucleotidase and aminopeptidase N, respectively. EGF did not influence cyclic AMP production, and its effect on 5'-nucleotidase was additive to that of forskolin or 8-bromo-cAMP. In contrast, genistein (10 mg x ml(-1)), an inhibitor of tyrosine kinase, prevented EGF-dependent stimulation of aminopeptidase N and 5'-nucleotidase, suggesting that protein phosphorylation was involved in the signaling mechanism. EGF stimulated specifically the latter two enzymes since it had no effect on other ectoenzymes including alkaline phosphodiesterase I and Mg2+-ATPase activities. These results demonstrate that EGF, via the control of 5'-nucleotidase and aminopeptidase N, which are implied in adenosine formation and peptide processing, respectively, could play a role in human cultured mesangial cell contractility and proliferation.
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PMID:Epidermal growth factor upregulates aminopeptidase N and 5'-nucleotidase in human glomerular mesangial cells. 985 17

The role of regucalcin, a regulatory protein in intracellular signaling system, in the regulation of Ca2+-ATPase activity in rat heart mitochondria was investigated. Mitochondrial Ca2+-ATPase activity was significantly increased by increasing concentrations of CaCl2 (2.5-50 microM). An increase in the enzyme activity was saturated at 50 microM CaCl2. The addition of regucalcin (10(-11)-10(-8) M) in the enzyme reaction mixture caused a significant increase in Ca2+-ATPase activity in heart mitochondria in the presence of 50 microM CaCl2. Regucalcin did not have a significant effect on mitochondrial Mg2+-ATPase activity. Regucalcin (10(-9) M) did not have a significant effect on Ca2+-ATPase activity in the presence of digitonin (10(-3) or 10(-2) %), which is a solubilization effect on membranous lipids. The effect of regucalcin in increasing mitochondrial Ca2+-ATPase activity was not observed in the presence of ruthenium red (10(-7) M) or lanthanum chloride (10(-7) M), which is an inhibitor of Ca2+ uniporter. The effect of regucalcin (10(-9) M) in increasing mitochondrial Ca2+-ATPase activity was not significantly enhanced in the presence of calmodulin (5 microg/ml) or dibutyryl cyclic AMP (10(-4) M), which is an intracellular signaling factor that can cause a significant increase in the enzyme activity. Mitochondrial regucalcin localization was significantly increased in the heart of regucalcin transgenic rats as compared with that of normal rats using Western blot analysis. Ca2+-ATPase activity was significantly increased in the heart mitochondria of regucalcin transgenic rats. This study demonstrates that regucalcin has an activating effect on Ca2+-ATPase in rat heart mitochondria, suggesting its role in the regulation of heart mitochondrial function.
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PMID:Regucalcin increases Ca2+-ATPase activity in the heart mitochondria of normal and regucalcin transgenic rats. 1678 69

The biguanide metformin is an oral antihyperglycemic drug for the treatment of type 2 diabetes mellitus. Further, a moderate improvement of dyslipidemia by metformin was reported, and therefore, the effect of metformin on the release of apolipoprotein B (ApoB) and ApoE in primary human hepatocytes was determined. Metformin at 0.5 and 1 mM reduced hepatic ApoB secretion but ApoE was not altered. Metformin is well known to stimulate the AMP kinase that subsequently reduces hepatic nuclear factor 4-alpha (HNF4-alpha) and HNF4-alpha regulated genes like ApoB. However, HNF4-alpha was only diminished by 1 mM metformin and ApoB mRNA was not suppressed indicating that this pathway may not explain reduced ApoB release. Lower abundance of lysophosphatidylcholine (lysoPC) may also diminish ApoB secretion. Therefore, electrospray ionization tandem mass spectrometry was applied to measure cellular lipids. PC, lysoPC (produced by hydrolysis of PC), phosphatidylserine and sphingomyelin (derived from PC) were lower in metformin-treated hepatocytes whereas phosphatidylethanolamine, an alternative precursor of PC, was not affected. In addition, ABCB4, the canalicular membrane flippase essential for biliary PC secretion, was diminished. Supplementation with lysoPC led to a selective elevation of endogenous lysoPC and rescued ApoB secretion in metformin-treated cells. Therefore, it is concluded that metformin reduces lysoPC in human hepatocytes and this may secondarily lead to a therapeutically beneficial lower release of ApoB.
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PMID:Metformin reduces cellular lysophosphatidylcholine and thereby may lower apolipoprotein B secretion in primary human hepatocytes. 1850 22


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