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)

Na+, K+-dependent ATPase [EC 3.6.1.3] was purified from porcine kidney by the method of Lane et al. [(1973) J. Biol. Chem. 248, 7197-7200] with slight modifications [Yamaguchi, M. & Tonomura, Y., (1979) J. Biochem. 86, 509-523]. The amounts of a phosphorylated intermediate (EP) and ouabain bound to the enzyme during the ATPase reaction were measured in 2.1 mM MgCl2 and various concentrations of NaCl and KCl at pH 7.5 and 20 degrees C. In presence of NaCl and the absence of KCl, the molar ratio of the amounts of EP and bound ouabain was 1 : 2. In the presence of both NaCl and KCl, it was 1 : 1. In both cases, the amount of bound ouabain was equal to that of EP in the absence of ouabain. These findings suggest that the functional unit of the transport ATPase is a dimer.
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PMID:Binding of ouabain to Na+, K+-dependent ATPase during the ATPase reaction. Evidence for a dimer structure of the ATPase. 22 43

Proteins of apparent molecular weights between 10 000 and 250 000 could be solubilized from guinea pig epidermis using a Tris/sucrose/ATP buffer. When the ionic concentration of the solubilized extract was made 75 mM with respect to KCl and 2 mM with respect to MgCl2, a protein complex precipitated which on SDS-polyacrylamide gel electrophoresis resolved into bands corresponding in migration to myosin, actin and a number of low molecular weight proteins. Myosin was dissociated from the complex with 0.6 M KI and purified by gel filtration chromatography on an agarose column. The purified epidermal myosin fraction contained a polypeptide of 200 000 molecular weight andtwo low molecular weight polypeptides of 16 500 and 13 000. The amino acid composition of the epidermal myosin heavy chain was similar to that of muscle myosin. At high ionic strength epidermal myosin had high specific (K+ + Ca2+)- and (K+ + EDTA)-ATPase activities and low specific (K+ + Mg2+)-ATPase activity. The pH activity curves of the (K+ + Ca2+)- and (K+ + EDTA)-ATPase were different. ATP was hydrolyzed faster than other nucleoside triphosphates. At low ionic strength, the (K+ + Mg2+)-ATPase activity of epidermal myosin was stimulated two fold by skeletal muscle actin. The myosin formed bipolar filaments in 50 mM KCl in the presence of 5 mM Mg2+.
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PMID:Contractile proteins in epidermis. Isolation and properties of guinea-pig epidermal myosin. 22 13

A large fraction of the Ca-2plus- and Mg-2plus-dependent ATPase (EC 3.6.1.3) in sarcoplasmic reticulum membranes solubilized with Triton X-100 was phosphorylated with Pi. The phosphorylation required Mg-2plus but was strongly inhibited by low concentrations of Ca-2plus. A Ca-2plus ion concentration of 30 muM caused half-maximum inhibition in the presence of 50 mM MgCl2. The phosphorylated enzyme showed a rapid turnover and was in dynamic equilibrium with Pi in the medium. At equilibrium the amount of the phosphorylated enzyme increased markedly with increased in the reaction temperature. The apparent standard free energy change, the apparent standard enthalpy change, and the apparent standard entropy change in the formation of the phosphorylated enzyme from the enzyme-phosphate complex in the presence of excess Mg-2plus at 37 degrees and pH 7.0 were, respectively, 0.35 Cal per mol, 15.9 Cal per mol, and 50.2 e.u. per mol. The susceptibility of the acid-denatured phosphorylated enzyme to hydroxylamine showed that the phosphorylated enzyme is of an acyl phosphate type. The present results are consistent with the probability that the phosphorylation results from reversal of late steps in the Ca-2plus transport process. The results clearly show that the phosphorylated enzyme is stabilized by a great increase in entropy upon its formation from the enzyme-phosphate complex.
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PMID:Phosphorylation of solubilized sarcoplasmic reticulum by orthophosphate and its thermodynamic characteristics. The dominant role of entropy in the phosphorylation. 23 82

Actomyosin was extracted from smooth muscle of molluscan abalone with 0.1 M PPit pH 6.4. Myosin was separated from the actomyosin by centrifugation at 100,000 X g in the presence of 5 mM ATP and 10 mM MgCl2. Myosin in the supernatant was further purified by gel filtration on a Sepharose 4B column. Paramyosin contamination of the actomyosin preparation interfered with the isolation of myosin and complete removal of actin and paramyosin from the myosin has not been accomplished. The myosin appeared to consist of a single f-chain and a single g-chain, as examined by SDS-disc electrophoresis in 8 or 13.7% acrylamide gel. The ATPase [EC 3.6.1.3] activity of this myosin in 0.5 M KCL at neutral pH and at 0 degrees was rather unstable and decreased by 10-20% per day. The effects of rho-chloromercuribenzoate and EDTA on the ATPase activity were similar to those observed with other smooth muscle myosin but the dependence upon pH or KCL concentration was different.
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PMID:Myosin from molluscan abalone, Haliotis discus. Isolation and enzymatic properties. 23 37

Efflux of Ca2+ from reversibly hemolyzed human red blood cells ghosts was determined by a Ca2+ selective electrode, by atomic absorption spectroscopy, and by the use of 45Ca. Hydrolysis of ATP was determined by measurement of inorganic phosphate (Pi). At 25 degrees C, ghosts loaded with CaCl2, MgCl2, Na2ATP, and Tris buffer (pH 7.4) extruded Ca2+, with mean rates ranging from 58.8 +/- 3.5 (SD) to 74.7 +/- 8.2 (SD) mumoles.liter ghosts -1.min-1 depending on the method of Ca2+ determination. The ratio of Ca2+ transport to Pi released in the presence of ouabain without correction for background ATP splitting was 0.83, 0.83, and 0.80, respectively, for the three methods of Ca2+ determination. Correction for the ATPase activity not associated with Ca2+ transport resulted in a ratio of 0.91:1. In other experiments, the use of La3+ to inhibit the Ca2+-pump allowed an estimate of the ATPase activity associated with Ca2+ extrusion. In the presence of various concentrations of La3+, the ratio of Ca2+ pumped to Pi liberated was 0.86 or 1.02, depending on the method of Ca2+ determination. It is concluded that the stoichiometry of the Ca2+-pump of the RBC plasma membrane is one Ca2+ pumped per ATP hydrolyzed.
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PMID:On the red blood cell Ca2+-pump: an estimate of stoichiometry. 69 Oct 40

Our previous studies have shown a salutary effect of adenosine triphosphate-magnesium chloride (ATP-MgCl2) administered to animals in shock. The presence of adenine nucleotide converting enzymes on cell surfaces and the ability of nucleotides to act at the cell surface have been recognized also. To investigate the fate of administered or externally applied ATP and to determine whether it would be subjected to increased degradation with shock, the soteus muscles from rats subjected to hemorrhagic shock and from control animals were incubated in the presence of ATP, adenosine diphosphate (ADP), or adenosine monophosphate (AMP) with MgCl2. Comparable degradation of the added nucleotides was observed with both control muscles and those from bled animals. Adenylate kinase activity was detected to the same extent in the medium after incubation with both groups of muscles, but other enzymes were not, suggesting that the latter enzymes were located on the exterior surface of the muscle cell. Thus with shock there was no increase in the breakdown of the nucleotides by the enzymes on the muscle surface (ATPase, AMPdeaminase) or the cellular enzyme, adenylate kinase.
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PMID:Degradation of adenine nucleotides by the soleus muscle in hemorrhagic shock. 112 91

Urea, in nondenaturing concentrations, inhibited Ca2+ uptake by sarcoplasmic reticulum vesicles with no concomitant effect on ATP hydrolysis. This inhibition was antagonized by 5 mM oxalate and 20 mM orthophosphate. At concentrations of 0.2 to 1.0 M, urea induced an increase in the Ca2+ efflux from preloaded vesicles diluted in a medium at pH 7.0 containing 2 mM ethylene glycol bis(beta-aminoethyl ether)N,N'-tetraacetic acid, 0.1 mM orthophosphate, and 0.1 mM MgCl2. The urea-induced efflux was arrested by ligands of the (Ca(2+)-Mg2+) ATPase, namely, K+, Mg2+, Ca2+, and ADP, and by ruthenium red and the polyamines spermine, spermidine, and putrescine. In the case of polyamines a dissociation between the effect on the efflux and the net Ca2+ uptake was observed, as only the efflux could be blocked by the drugs. Glycine betaine, trimethylamine-N-oxide, and sucrose antagonized the effects of urea on both the net Ca2+ uptake and the rate of Ca2+ efflux.
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PMID:The enhancement of Ca2+ efflux from sarcoplasmic reticulum vesicles by urea. 128 64

We have investigated several purification strategies for the cystic fibrosis transmembrane regulator (CFTR) based on its structural similarity to other proteins of the traffic ATPase/ABC transporter family. Recombinant CFTR expressed in heterologous cells was readily solubilized by digitonin and initially separated from the majority of other cellular proteins by sucrose density gradient centrifugation. CFTR, with two predicted nucleotide binding domains, bound avidly to several triazine dye columns, although elution with MgATP, MgCl2, or high ionic strength buffers was inefficient. CFTR did not bind to either ATP or ADP coupled to agarose. Because CFTR is a glycoprotein we investigated its binding to lectin columns. CFTR bound readily to wheat germ agglutinin, but poorly to Lens culinaris agglutinin. CFTR was enriched 9-10 times when eluted from wheat germ agglutinin with N-acetylglucosamine. This enrichment was tripled if lectin chromatography followed sucrose gradient centrifugation. Our results suggest the combination of sucrose density gradient centrifugation and lectin chromatography would be a satisfactory approach to initial purification of CFTR expressed in heterologous cells.
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PMID:Partial purification of the cystic fibrosis transmembrane conductance regulator. 128 84

We have used liposomes with incorporated pig kidney Na+,K(+)-ATPase to study vanadate sensitive K(+)-K+ exchange and net K+ uptake under conditions of acetyl- and p-nitrophenyl phosphatase activities. The experiments were performed at 20 degrees C. Cytoplasmic phosphate contamination was minimized with a phosphate trapping system based on glycogen, phosphorylase a and glucose-6-phosphate dehydrogenase. In the absence of Mg2+ (no phosphatase activity) 5-10 mM p-nitrophenyl phosphate slightly stimulated K(+)-K+ exchange whereas 5-10 mM acetyl phosphate did not. In the presence of 3 mM MgCl2 (high rate of phosphatase activity) acetyl phosphate did not affect K(+)-K+ exchange whereas p-nitrophenyl phosphate induced a greater stimulation than in the absence of Mg2+; a further addition of 1 mM ADP resulted in a 35-65% inhibition of phosphatase activity with an increase in K(+)-K+ exchange, which sometimes reached the levels seen with 5 mM phosphate and 1 mM ADP. The net K+ uptake in the presence of 3 mM MgCl2 was not affected by acetyl phosphate or p-nitrophenyl phosphate, whereas it was inhibited by 5 mM phosphate (with and without 1 mM ADP). The results of this work suggest that the phosphatase reaction is not by itself associated to K+ translocation. The ADP-dependent stimulation of K(+)-K+ exchange in the presence of phosphatase activity could be explained by the overlapping of one or more step/s of the reversible phosphorylation from phosphate with the phosphatase cycle.
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PMID:Phosphatase activity and potassium transport in liposomes with Na+,K(+)-ATPase incorporated. 130 62

The aim of the present work was to elucidate the role played by ATP and Mg2+ ions in the early steps of the Na+,K(+)-ATPase cycle. The approach was to follow pre-steady-state phosphorylation kinetics in Na(+)-containing K(+)-free solutions under variable ATP and MgCl2 concentrations. The experiments were performed with a rapid mixing apparatus at 20 +/- 2 degrees C. The concentrations of free and complexes species of Mg2+ and ATP were calculated on the basis of a dissociation constant of 0.091 +/- 0.004 mM, estimated with Arsenazo III under identical conditions. A simplified scheme were ATP binds to the ENa enzyme, which is phosphorylated to MgEPNa and consequently dephosphorylated returning to the ENa form, was used. In the absence of ADP and phosphate four rate constants are relevant: k1 and k-1, the on and off rate constants for ATP binding; k2, the transphosphorylation rate constant and k3, the constant that governs the dephosphorylation rate. The values obtained were: k1 = 0.025 +/- 0.003 microM-1 ms-1 for both free ATP and ATPMg; k-1 = 0.038 +/- 0.004 ms-1 for free ATP and 0.009 +/- 0.002 ms-1 for ATPMg; k2 = 0.199 +/- 0.005 ms-1; k3 = 0.0019 +/- 0.0002 ms-1. The model that seems best to explain the data is one where (i) the role of true substrate can be played equally well by free ATP or ATPMg, and (ii) free Mg2+, an essential activator, acts by binding to a specific Mg2+ site on the enzyme molecule.
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PMID:Effects of magnesium and ATP on pre-steady-state phosphorylation kinetics of the Na+,K(+)-ATPase. 131 73


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