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)

1. The effect of free Mg2+, MgEDTA and MgCDTA on the phofphorylation of the (Na+ + K+)-activated ATPase (ATP phosphohydrolase, EC 3.6.1.3) has been studied. 2. 10 mM trans-1,2-diaminocyclohexane-N,N,N',N'-tetraacetic acid (CDTA) added simultaneously with [gamma-32P]ATP to a solution containing the enzyme, 1 mM Mg2+ and 150 mM Na+ does not prevent formation of phospho-enzyme. When [gamma-32P]ATP is added after CDTA the level of phospho-enzyme obtained decreases with increase in the time interval between addition of CDTA and ATP. The inability of CDTA to prevent the formation of phospho-enzyme becomes more pronounced when the medium contains MgEDTA. In the presence of CDTA the maximum amount of phospho-enzyme formed increases with the MgEDTA concentration. 3. Without CDTA the steady-state level of phospho-enzyme is directly proportional to the logarithm of free Mg2+ concentration. Neither with suboptimal nor with optimal concentrations of free Mg2+ does MgEDTA have an effect on the level of phospho-enzyme formed. 4. Using the phospho-enzyme level as a measure of free Mg2+ the experiments show that CDTA reacts slower with Mg2+ than does EDTA, but the stability constant of MgCDTA complex is higher than of MgCDTA, complex. 5. Due to the higher stability constant, of MgCDTA, as compared to MgEDTA, addition of CDTA to a medium containing free Mg2+ and MgEDTA will not only chelate the free Mg2+, but it will also shift the equilibrium from MgEDTA towards MgCDTA, i.e. MgEDTA acts as a source of free Mg2+ which is then chelated by CDTA. The experiments show that it takes minutes before Mg2+, EDTA and CDTA come to equilibrium. Provided the dissociation of MgEDTA is faster than the formation of the MgCDTA complex, the medium will contain a concentration of free Mg2+ which at any given instant is near in equilibrium with a slowly decreasing concentration of MgEDTA; this free Mg2+ can support phosphorylation. This can explain why the rate with which CDTA stops phosphorylation decreases with an increase in the MgEDTA concentration. 6. When phosphorylation is stopped by addition of unlabelled ATP, the rate of dephosphorylation is faster than when it is stopped by addition of CDTA both with and without EDTA in the medium. CDTA reacts too slowly with Mg2+ to be used as a chelator in studies where a fast removal of Mg2+ is required. 7. A previous finding has been verified, namely that the rate of spontaneous, of K+-stimulated and of ADP-stimulated dephosphorylation is independent of the Mg2+ concentration during formation of phospho-enzyme.
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PMID:The effect of chelators on Mg2+, Na+-dependent phosphorylation of (Na+ + K+)-activated ATPase. 13 34

Troponin C was isolated from the skeletal muscle of bullfrog (Rana catesbeiana), and its relative molecular mass was estimated to be 18,000 by SDS/polyacrylamide gel electrophoresis. In its amino acid composition, bullfrog troponin C was similar to that of the frog (Rana esculenta) but different from that of rabbit. Its ultraviolet spectrum was consistent with its amino acid composition. The ultraviolet difference spectrum of the Ca(2+)-loaded form vs. the metal-free form indicated that the single Tyr residue and some Phe residues in the bullfrog troponin C molecule were affected by the conformational change associated with Ca2+ binding. On electrophoresis in polyacrylamide gel in 14 mM Tris and 90 mM glycine, the metal-free and Mg(2+)-loaded forms migrated slower than the Ca(2+)-loaded form. The property is shared by rabbit troponin C but not parvalbumins or calmodulin. The ATPase activity of CDTA-treated myofibrils reconstituted with bullfrog troponin C showed the same Ca(2+)- and Sr(2+)-sensitivity as that of those reconstituted with rabbit troponin C. Bullfrog troponin C is, thus, physiologically the same as rabbit troponin C, in spite of several marked differences in their physicochemical properties.
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PMID:Preparation and characterization of troponin C from bullfrog skeletal muscle. 129 89

The effect of Mg2+ on the Ca2+ binding to rabbit fast skeletal troponin C and the CA2+ dependence of myofibrillar ATPase activity was studied in the physiological state where troponin C was incorporated into myofibrils. The Ca2+ binding to troponin C in myofibrils was measured directly by 45Ca using the CDTA-treated myofibrils as previously reported (Morimoto, S. and Ohtsuki, I. (1989) J. Biochem. 105, 435-439). It was found that the Ca2+ binding to the low and high affinity sites of troponin C in myofibrils was affected by Mg2+ competitively and the Ca2(+)- and Mg2(+)-binding constants were 6.20 x 10(6) and 1.94 x 10(2) M-1, respectively, for the low affinity sites, and 1.58 x 10(8) and 1.33 x 10(3) M-1, respectively, for the high affinity sites. The Ca2+ dependence of myofibrillar ATPase was also affected by Mg2+, with the apparent Ca2(+)- and Mg2(+)-binding constants of 1.46 x 10(6) and 276 x 10(2) M-1, respectively, suggesting that the myofibrillar ATPase was modulated through a competitive action of Mg2+ on Ca2+ binding to the low affinity sites, though the Ca2+ binding to the low affinity sites was not simply related to the myofibrillar ATPase.
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PMID:The effect of Mg2+ on the Ca2+ binding to troponin C in rabbit fast skeletal myofibrils. 182 17

We have developed a procedure to discriminate actomyosin-type ATPase activity from Ca(2+)-ATPase activity of sarcoplasmic reticulum (SR) in mechanically skinned fibres, determining simultaneously their Ca(2+)-induced tension and accompanying ATPase activity. When they were treated with an alkaline CyDTA-containing solution of low ionic strength which was reported to remove troponin C, the fibres showed a considerable amount of Ca(2+)-dependent ATPase activity, in spite of having little or no Ca(2+)-induced isometric tension. The residual ATPase activity is ascribed to the Ca(2+)-ATPase activity of SR, because it is completely abolished by 1% CHAPS treatment for 10 min. This conclusion is also supported by the finding that the Ca(2+)-dependence of the ATPase activity is very similar to that of Ca(2+)-ATPase of SR isolated from rabbit skeletal muscle, and that the estimated activity is consistent with the reported values of direct determinations. On the other hand, treatment with a detergent such as CHAPS or Triton X-100 removes SR activities (ATPase and Ca-uptake), leaving Ca(2+)-induced tension and actomyosin-type ATPase activity unchanged. This procedure indicated that the contribution of Ca(2+)-ATPase activity of SR may be minimal in total steady-state ATPase activity of mechanically skinned mammalian skeletal muscle fibres. Successive CyDTA and CHAPS treatments eliminated both Ca(2+)-induced tension and ATPase activity, which were recovered by the addition of troponin C. Using these procedures, we also examined the effect of cyclopiazonic acid (CPA) which was reported to be a specific inhibitor of Ca(2+)-ATPase of SR. Ca(2+)-ATPase activity of SR in skinned fibres was inhibited completely by 10 microM CPA and held to one-half by about 0.2 microM. This effect was only partially reversible. CPA at 10 microM or higher concentrations showed Ca(2+)-sensitizing action on myofibrils, which was readily reversible. CPA at 3 microM inhibited almost completely the Ca(2+)-ATPase activity of SR, while it had no effect on either actomyosin-type ATPase or isometric tension of myofibrils.
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PMID:Discrimination of Ca(2+)-ATPase activity of the sarcoplasmic reticulum from actomyosin-type ATPase activity of myofibrils in skinned mammalian skeletal muscle fibres: distinct effects of cyclopiazonic acid on the two ATPase activities. 183 95

The plasma membrane fraction of chicken osteoclasts was purified utilizing 20% continuous Percoll gradients. Biochemical marker enzyme analysis (ouabain-sensitive Na+,K(+)-ATPase and 5'-nucleotidase) indicated that plasma membrane enrichment was 11.87-fold and 7.25-fold, respectively, and contamination with mitochondria, endoplasmic reticulum, and lysosomes was low as determined by succinic dehydrogenase, NADH dehydrogenase, and N-acetylglucosaminidase activities, respectively. SDS latency of Na+,K(+)-ATPase and 5'-nucleotidase activities of the isolated plasma membranes revealed that 43-50% of vesicles were sealed, with 10-16% in the inside-out orientation, depending on the membrane fraction used. Electron microscopy confirmed the vesicular nature of the plasma membrane fraction. The plasma membrane Ca2(+)-ATPase had a high-affinity (KCa = 0.22 microM; Vmax = 0.16 mumol/mg per min) and a low-affinity (KCa = 148 microM; Vmax = 0.37 mumol/mg per min) component. Calmodulin (0.12 microM) had no effect on Ca2(+)-ATPase activity. However, trifluoperazine (0.1 mM), a calmodulin antagonist, strongly inhibited especially the high-affinity component of the enzyme. Vanadate and lanthanum also caused inhibition. In the presence of CDTA, a potent Ca2+ and Mg2+ chelating agent, high-affinity Ca2(+)-ATPase activity was abolished, indicating that trace Mg2+ was essential for activity. The Ca2(+)-ATPase substrate curve using ATP showed a high-affinity (Km = 12.3 microM; Vmax = 0.022 mumol/mg per min) and a low-affinity (Km = 43.8 microM; Vmax = 0.278 mumol/mg per min) component. These results demonstrate that osteoclasts have a plasma membrane Ca2(+)-ATPase with characteristics similar to the enzyme responsible for active calcium extrusion in other cells.
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PMID:Characterization of a Ca2(+)-ATPase in osteoclast plasma membrane. 214 47

ATPase activity in rat heart sarcoplasmic reticulum was stimulated in a concentration-dependent manner by both Ca2+ and Mg2+ in the complete absence of the other cation. Increasing concentrations of Mg2+ produced an apparent inhibition of the Ca2(+)-dependent ATP hydrolysis. CDTA (trans-1,2-diaminocyclo-hexane-N,N,N',N'-tetraacetate) had no effect on these responses. The results indicate the presence of a low affinity non-specific divalent cation-stimulated ATPase in rat heart sarcoplasmic reticulum. However, sarcoplasmic reticulum vesicles transported Ca2+ with a high affinity (K0.5 Ca2+ = 0.41 microM) suggesting the presence of a high affinity Ca2(+)-transporting ATPase. Calmodulin did not stimulate rat heart sarcoplasmic reticulum ATPase activity over a range of Ca2+ and Mg2+ concentrations and failed to stimulate membrane phosphorylation and Ca2+ transport into sarcoplasmic reticulum vesicles. Calmodulin antagonists trifluoperazine and compound 48/80 did not affect the ATPase activity. Catalytic subunit of cAMP-dependent protein kinase was also ineffective in stimulating the ATPase activity. These results suggest the presence of an ATPase activity in rat heart sarcoplasmic reticulum with different properties from the high affinity Ca2(+)-pumping ATPase previously characterized in dog heart and other species.
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PMID:A non-specific Ca2+ (or Mg2+)-stimulated ATPase in rat heart sarcoplasmic reticulum. 214 1

Ca2+ binding to skeletal muscle troponin C in skeletal or cardiac myofibrils was measured by the centrifugation method using 45Ca. The specific Ca2+ binding to troponin C was obtained by subtracting the amount of Ca2+ bound to the CDTA-treated myofibrils (troponin C-depleted myofibrils) from that to the myofibrils reconstituted with troponin C. Results of Ca2+ binding measurement at various Ca2+ concentrations showed that skeletal troponin C had two classes of binding sites with different affinity for Ca2+. The Ca2+ binding of low-affinity sites in cardiac myofibrils was about eight times lower than that in skeletal myofibrils, while the high-affinity sites of troponin C in skeletal or cardiac myofibrils showed almost the same affinity for Ca2+. The Ca2+ sensitivity of the ATPase activity of skeletal troponin C-reconstituted cardiac myofibrils was also about eight times lower than that of skeletal myofibrils reconstituted with troponin C. These findings indicated that the difference in the sensitivity to Ca2+ of the ATPase activity between skeletal and cardiac CDTA-treated myofibrils reconstituted with skeletal troponin C was mostly due to the change in the affinity for Ca2+ of the low-affinity sites on the troponin C molecule.
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PMID:Ca2+ binding to skeletal muscle troponin C in skeletal and cardiac myofibrils. 252 23

CDTA-treatment of rabbit skeletal natural actomyosin resulted in the complete removal of troponin C and the abolishment of Ca2+-activated ATPase activity. The addition of troponin C restored the Ca2+-activated ATPase activity to almost the same extent as that of the intact natural actomyosin. These finding are consistent with those of previous studies on the skeletal and cardiac myofibrils.
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PMID:Removal of troponin C from natural actomyosin of rabbit skeletal muscle by CDTA. 252 70

We have developed a new method to prepare single-headed heavy meromyosin with high purity and a high yield. To examine whether the two heads on the same myosin molecule work cooperatively or not, it is important to prepare pure single-headed heavy meromyosin. Myosin was extracted from myofibrils treated with a solution containing CyDTA, a strong divalent cation chelator. CyDTA treatment was essential to the production of sHMM. Then such myosin was digested with chymotrypsin in the presence of divalent cations at high ionic strength. Crude sHMM was separated from double-headed HMM by affinity chromatography using an ADP-column. Contaminating S1 was removed by gel filtration. Heavy chain of sHMM obtained by the present method had no nick. Purified sHMM showed normal EDTA-ATPase and Ca-ATPase. It interacted with thin filament and its ATPase was activated by actin normally.
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PMID:New method to prepare single-headed heavy meromyosin with high purity and a high yield. 253 47

In order to determine the role of divalent cations in the reaction mechanism of the H+,K+-ATPase, we have substituted calcium for magnesium, which is required by the H+,K+-ATPase for phosphorylation from ATP and from PO4. Calcium was chosen over other divalent cations assayed (barium and manganese) because in the absence of magnesium, calcium activated ATP hydrolysis, generated sufficiently high levels of phosphoenzyme (573 +/- 51 pmol.mg-1) from [gamma-32P]ATP to study dephosphorylation, and inhibited K+-stimulated ATP hydrolysis. The Ca2+-ATPase activity of the H+,K+-ATPase was 40% of the basal Mg2+-ATPase activity. However, the Ca2+,K+-ATPase activity (minus the Ca2+ basal activity) was only 0.7% of the Mg2+,K+-ATPase, indicating that calcium could partially substitute for Mg2+ in activating ATP hydrolysis but not in K+ stimulation of ATP hydrolysis. Approximately 0.1 mM calcium inhibited 50% of the Mg2+-ATPase or Mg2+,K+-ATPase activities. Inhibition of Mg2+,K+-ATPase activity was not competitive with respect to K+. Inhibition by calcium of Mg2+,K+ activity p-nitrophenyl phosphatase activity was competitive with respect to Mg2+ with an apparent Ki of 0.27 mM. Proton transport measured by acridine orange uptake was not detected in the presence of Ca2+ and K+. In the presence of Mg2+ and K+, Ca2+ inhibited proton transport with an apparent affinity similar to the inhibition of the Mg2+, K+-ATPase activity. The site of calcium inhibition was on the exterior of the vesicle. These results suggest that calcium activates basal turnover and inhibits K+ stimulation of the H+,K+-ATPase by binding at a cytosolic divalent cation site. The pseudo-first order rate constant for phosphoenzyme formation from 5 microM [gamma-32P]ATP was at least 22 times slower in the presence of calcium (0.015 s-1) than magnesium (greater than 0.310 s-1). The Ca.EP (phosphoenzyme formed in the presence of Ca2+) formed dephosphorylated four to five times more slowly that the Mg.EP (phosphoenzyme formed in the presence of Mg2+) in the presence of 8 mm trans-1,2-diaminocyclohexane-N,N,N',N'-tetraacetic acid (CDTA) or 250 microM ATP. Approximately 10% of the Ca.EP formed was sensitive to a 100 mM KCl chase compared with greater than 85% of the Mg.EP. By comparing the transient kinetics of the phosphoenzyme formed in the presence of magnesium (Mg.EP) and calcium (Ca.EP), we found two actions of divalent cations on dephosphorylation.(ABSTRACT TRUNCATED AT 400 WORDS)
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PMID:The substitution of calcium for magnesium in H+,K+-ATPase catalytic cycle. Evidence for two actions of divalent cations. 255 12


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