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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 capacity of various ATPase preparations from beef heart mitochondria to catalyze exchange of phosphate oxygens with water has been evaluated. Oligomycin-sensitive ATPase preparations retain a capacity for considerable intermediate Pi equilibrium HOH exchange per Pi formed during ATP hydrolysis at relatively high ATP concentration (5 mM). Submitochondrial particles prepared by an ammonia-Sephadex procedure with 5 mM ATP showed more rapid ATPase, less oligomycin sensitivity, and less capacity for intermediate exchange. With these particles, intermediate Pi equilibrium HOH exchange per Pi formed was increased as ATP concentration was decreased. The purified, soluble ATPase from mitochondria catalyzed little or no intermediate Pi equilibrium HOH exchange at 5 mM ATP but showed pronounced increase in capacity for such exchange as ATP concentration was lowered. The ATPase also showed a weak catalysis of an ADP-stimulated medium Pi equilibrium HOH exchange. The results support the alternating catalytic site model for ATP synthesis or cleavage. They also demonstrate that a transmembrane protonmotive force is not necessary for oxygen exchange reactions. At lower ATP concentrations, ADP and Pi formed at a catalytic site appear to remain bound and continue to allow exchange of Pi oxygens until ATP binds at another site on the enzyme.
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PMID:Occurrence and significance of oxygen exchange reactions catalyzed by mitochondrial adenosine triphosphatase preparations. 15 10

The Mg2+- and Ca2+-stimulated ATPase (bacterial coupling factor) has been investigated in solution with different independent techniques. The molecular weight of the five-subunit enzyme was found to be 345,000 +/- 5,000 by means of light scattering, 350,000 by sedimentation equilibrium experiments, and 358,000 by means of small-angle x-ray scattering. The radius of gyration was found to be 41.9 A, the volume 7.39 x 10(5) A3, and the surface to volume ratio 5.5 x 10(-2) A-1 from small-angle x-ray scattering measurements of the enzyme in solution. The degree of hydration was found to be 0.62 ml of H2O/g of ATPase. The translational diffusion coefficient was determined to be 3.47 x 10(-7) cm2 s-1 by means of inelastic light scattering. The distribution of the scattered intensity near the origin appears to be bimodal, suggesting that the ATPase molecule is composed of spherical parts bound together by a flexible polypeptide chain. The largest dimension of the ATPase in solution is 120.0 A, determined from the pair distribution function.
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PMID:Size and molecular parameters of adenosine triphosphatase from Escherichia coli. 15 80

In experiments on isolated gallbladders (GB) of frogs it was established that noradrenaline in concentration of 6.10(-9)--3.10(-4) M acting on the organ from the serosalsurface causes firstly a short increase and then -- a prolonged inhibition of the absorption rate of NaCl -- isotonic fluid from the gall bladder cavity. While the concentration of the mediator increases in the inculation medium, its inhibiting effect increases too. Depression, and at high concentration in the medium, full inhibition of the process of fluid absorption is accompanied with distinct decrease of Na--, K--ATPase activity of gall bladder epithelial cells. Mediator in concentration of 3.10(-8) M caused an increase of membrane potential of the epithelial cells, while its increasing in the inculation medium to 3.10(-6) M caused a decrease of the transmembrane potential difference. Under the noradrenaline influence the increase of the osmotic permeability of the gall bladder's wall for the water flow directed from the mucosa to the serosalsurface of the organ took place, and also the decrease of the wall's permeability for the water flow in the opposite direction was seen. It was concluded that the noradrenaline inhibitory action on the process of absorption of NaCl--isotonic fluid from the gall bladder cavity was observed because of the decrease of the Na--K--ATPase activity, and also because of the change of the permeability of epithelium for the passive ion and water transport.
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PMID:[Effect of noradrenaline on ion and water transport through frog gall bladder epithelium]. 15 53

We tested the hypothesis that membrane vesicles of smooth muscle function as organelles controlling cell volume through a mechanochemical mechanism not involving Na+-K+ dependent membrane ATPase. Pieces of rat myometrium were incubated under various conditions at 25 degrees C, and then were analyzed after various times for Na+, K+, ATP and water contents or were prepared and examined in the electron microscope. Metabolic inhibition with iodoacetate (IAA) + dinitrophenol (DNP) rapidly depleted ATP, then decreased membrane vesicle number and increased vesicle size. Thereafter K+ loss, Na+ gain and water gain occurred. Slower depletion of ATP by treatment of tissues with IAA or ethacrynic acid produced similar, but delayed effects. Treatment with DNP alone, DNP in glucose-free Krebs-Ringer or glucose-free solution bubbled with N2 partly depleted the tissues of ATP but did not markedly affect the membrane vesicles or tissue water content. Ouabain affected neither ATP contents of tissues nor the numberof membrane vesicles, but produced large intracellular vesicles. The membrane vesicles were suggested to be sites of a mechanochemical volume control system.
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PMID:Relation of membrane vesicles to volume and Na+ transport in smooth muscle: effect of metabolic and transport inhibition on fresh tissues. 15 83

Correlation between operation of a volume control system (independent of Na+-K+ -ATPase) and membrane vesicle structure was examined in strips of rat myometrium made Na-rich by incubation in Krebs-Ringer solutions at 5 degrees C. Some pieces of tissue were analyzed for Na+, K+, ATP and water contents before and after rewarming to 37 degrees C. Other pieces of tissue were prepared and examined in the electron microscope. The membrane vesicles along the surface of the muscle cells were reduced from the numberin fresh tissues made Na+-rich by overnight incubation in K+-free Ringer solution at 5 degrees C. Rewarming of Na+-rich tissues under conditions thought to inhibit Na+-K+-ATPase caused the loss of Na+ with water and with utake of K+ but the vesicles did not increase in number. Na+-K+ATPase activity was suggested to inhibit vesicle formation by competing for ATP. The loss of water and increase in vesicle number was Ca2+-dependent but did not require Na+. Inhibition of the volume control system by iodoacetamide, Ca2+-free solution and other procedures resulted in swelling of membrane vesicles. The membrane vesicles were suggested to be sites of a mechanochemical system of volume control and the mechanochemical step was suggested to involve discharge of membrane vesicles.
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PMID:Relation of membrane vesicles to volume control and Na+-transport in smooth muscle: studies on Na+-rich tissues. 15 84

Calcium loading of skeletal muscle sarcoplasmic reticulum performed passively by incubation with high calcium concentrations (0.5--15 mM) on ice gives calcium loads of 50--60 nmol/mg sarcoplasmic reticulum protein. This accumulated calcium is not released by EGTA [ethyleneglycol bis-(2-aminoethyl)-N,N,N',N'-tetraacetic acid], but almost completely released by ionophore X-537A plus EGTA or phospholipase A plus EGTA treatment and is therefore assumed to be inside the sarcoplasmic reticulum. This calcium is distributed in one saturable and one non-saturable calcium compartment, as derived from the dependence of the calcium load on the calcium concentration in the medium. These compartments are assigned to bound and ionized calcium inside the sarcoplasmic reticulum, respectively. Maximum calcium binding under these conditions was 33 nmol/mg protein with an apparent half-saturation constant of 5,8 nmol/mg free calcium inside, or between 1.2 and 0.6 mM free calcium inside, assuming an average vesicular water space of 5 or 10 microliter/mg protein, respectively. Calcium-dependent phosphorylation of sarcoplasmic reticulum calcium-transport ATPase from orthophosphate depends on the square of free calcium inside, whilst inhibition of phosphorylation depends on the square of free calcium in the medium. Calcium-dependent phosphorylation appears to be determined by the free calcium concentrations inside or outside allowing calcium binding to the ATPase according to the two classes of calcium binding constants for low affinity calcium binding or high affinity calcium binding, respectively. It is further suggested that the saturation of the low-affinity calcium-binding sites of the ATPase facing the inside of the sarcoplasmic reticulum membrane is responsible for the greater apparent orthophosphate and magnesium affinity in calcium-dependent phosphorylation than in calcium-independent phosphorylation from orthophosphate. Maximum calcium-dependent phosphoprotein formation at 20 degrees C and pH 7.0 is about 4 nmol/mg sarcoplasmic reticulum protein.
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PMID:Ionized and bound calcium inside isolated sarcoplasmic reticulum of skeletal muscle and its significance in phosphorylation of adenosine triphosphatase by orthophosphate. 15 75

An ATPase complex sensitive to the energy transfer inhibitors oligomycin, dicyclohexylcarbodiimide and venturicidin has been solubilized from Rhodospirillum rubrum chromatophores with Triton X-100 and further purified by centrifugation on a glycerol gradient. The partially purified RrFo . F1 contains 13 distinct polypeptide subunits, as revealed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, including the subunits of the oligomycin-sensitive, water-soluble RrF1 ATPase. The ATPase activity of RrF0 . F1 as that of the membrane-bound enzyme complex depends on Ca2+ or Mg2+ and from detailed kinetic studies it is concluded that the divalent cation-ATP complex is the substrate for both ATPase complexes. Free ATP and free Mg2+ act as competitive inhibitors, with Ki values of 1 mM and 7 muM, respectively. The subunit composition of the purified RrFo . F1 and its similarity to the membrane-bound ATPase with respect to cation dependence and sensitivity to energy transfer inhibitors suggests that it contains all the subunits of the R. rubrum coupling factor-ATPase complex.
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PMID:Coupling factor ATPase complex of Rhodospirillum rubrum. Purification and characterization of an oligomycin and N,N'-dicyclohexylcarbodiimide-sensitive (Ca+ + Mg2+)-ATPase. 15 83

ATP concentration modulates oxygen exchange catalyzed by purified, soluble mitochondrial ATPase during ATP hydrolysis so that water oxygen incorporation into each Pi formed increases markedly as ATP concentration is lowered. This behavior is readily explained by catalytic cooperativity between subunits of the ATPase. However, other reasonable explanations also need consideration. A new approach for assessing these various explanations is used, based on measurement of the [18O]Pi species formed by hydrolysis of ATP highly labeled with 18O in the gamma-phosphoryl group. The results and other supporting data give what appears to be the most compelling evidence yet attained for alternating site catalytic cooperativity in an enzymic catalysis.
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PMID:Subunit interaction during catalysis. Alternating site cooperativity of mitochondrial adenosine triphosphatase. 15 96

(1) Photophosphorylation, Ca2+-ATPase and Mg2+-ATPase activities of isolated chloroplasts were inhibited 55--65% when the chemical potential of water was decreased by dehydrating leaves to water potentials (psi w) of --25 bars before isolation of the plastids. The inhibition could be reversed in vivo by rehydrating the leaves. (2) These losses in activity were reflected in coupling factor (CF1) isolated from the leaves, since CF1 from leaves with low psi w had less Ca2+-ATPase activity than control CF1 and did not recouple phosphorylation in CF1-deficient chloroplasts. In contrast, CF1 from leaves having high psi w only partially recoupled phosphorylation by CF1-deficient chloroplasts from leaves havig low psi w. This indicated that low psi w affected chloroplast membranes as well as CF1 itself. (3) Coupling factor from leaves having low psi w had the same number of subunits, and the same electrophoretic mobility, and could be obtained with the same yields as CF1 from control leaves. However, direct measurements of fluorescence polarization, ultraviolet absorption, and circular dichroism showed that CF1 from leaves having low psi w differed from control CF1. The CF1 from leaves having low psi w also had decreased ability to bind fluorescent nucleotides (epsilon-ATP and epsilon-ADP). (4) Exposure of isolated CF1 to low psi w in vitro by preincubation in sucrose-containing media inhibited the Ca2+-ATPase activity of the protein in subsequent assays without sucrose. Inclusion of 5 or 10 mM Mg2+ in the preincubation medium markedly inhibited Ca2+-ATPase activity. (5) These results show that CF1 undergoes changes in cells which alter its phosphorylating ability. Since low cell psi w changed the spectroscopic properties but not other protein properties of CF1, the changes were most likely caused by altered confurn, photophosphorylation. The inhibition of ATPase activity in CF1 in vitro at low psi w and high ion concentration mimicked the change in activity seen in vivo.
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PMID:Conformation and activity of chloroplast coupling factor exposed to low chemical potential of water in cells. 15 20

EPR and water proton relaxation rate (1/T1) studies of partially (40%) and "fully" (90%) purified preparations of membrane-bound (Na+ + K+) activated ATPase from sheep kidney indicate one tight binding site for Mn2+ per enzyme dimer, with a dissociation constant (KD = 0.88 muM) in agreement with the kinetically determined activator constant, identifying this Mn2+-binding site as the active site of the ATPase. Competition studies indicate that Mg2+ binds at this site with a dissociation constant of 1 mM in agreement with its activator constant. Inorganic phosphate and methylphosphonate bind to the enzyme-Mn2+ complex with similar high affinities and decrease 1/T1 of water protons due to a decrease from four to three in the number of rapidly exchanging water protons in the coordination sphere of enzyme-bound Mn2+. The relative effectiveness of Na+ and K+ in facilitating ternary complex formation with HPO2-4 and CH3PO2-3 as a function of pH indicates that Na+ induces the phosphate monoanion to interact with enzyme-bound Mn2+. Thus protonation of an enzyme-bound phosphoryl group would convert a K+-binding site to a Na+-binding site. Dissociation constants for K+ and Na+, estimated from NMR titrations, agreed with kinetically determined activator constants of these ions consistent with binding to the active site. Parallel 32Pi-binding studies show negligible formation (less than 7%) of a covalent E-P complex under these conditions, indicating that the NMR method has detected an additional noncovalent intermediate in ion transport. Ouabain, which increases the extent of phosphorylation of the enzyme to 24% at pH 7.8 and to 106% at pH 6.1, produced further decreases in 1/T1 of water protons. Preliminary 31P- relaxation studies of CH3PO2-3 in the presence of ATPase and Mn2+ yield an Mn to P distance (6.9 +/- 0.5 A) suggesting a second sphere enzyme-Mn-ligand-CH3PO2-3 complex. Previous kinetic studies have shown that T1+ substitutes for K+ in the activation of the enzyme but competes with Na+ at higher levels. From the paramagnetic effect of Mn2+ at the active site on the enzyme on I/T1 of 205T1 bound at the Na+ site, a Mn2+ to T1+ distance of 4.0 +/- 0.1 A is calculated, suggesting the sharing of a common ligand atomy by Mn2+ and T1+ on the ATPase. Addition of Pi increases this distance to 5.4 A consistent with the insertion of P between Mn2+ and T1+. These results are consistent with a mechanism for the (Na+ + K+)-ATPase and for ion transport in which the ionization state of Pi at a single enzyme active site controls the binding and transport of Na+ and K+, and indicate that the transport site for monovalent cations is very near the catalytic site of the ATPase. Our mechanism also accounts for the order of magnitude weaker binding of Na+ compared to K+.
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PMID:Magnetic resonance and kinetic studies of the mechanism of membrane-bound sodium and potassium ion- activated adenosine triphosphatase. 17 21


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