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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)

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

This paper reports on 1H and 31P NMR as well as EPR measurements of the labeling reagent of ATPase sites, "Co(III)-(phen)-ATP." This complex is found to be paramagnetic, as deduced both from its EPR spectrum and from the significant broadening, though almost unshifted, proton and phosphorus resonances. This paramagnetism is a result of the incorporation of the superoxide free-radical anion in the coordination sphere of the trivalent cobalt ion. Evidence for the presence of superoxide in the complex is based on competition experiments with cyanide, which is able to displace the superoxide anion. The latter was identified by its inducing effect on the photoreactivity of luminol. The displacement of superoxide by cyanide was accompanied by the abolition of the paramagnetism of the complex. The relative distances between the protons and phosphorus atoms of ATP and the superoxide anion in the complex were calculated using the NMR line-broadening data. Structural models compatible with the experimental results are proposed. Under conditions of excess of adenine nucleotides or phenanthroline, the coordinated ATP molecule becomes exchangeable. This phenomenon is attributed to the labilization of the cobaltic ion ligands induced by the superoxide anion.
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PMID:Structural and exchange properties of "Co(III)-phenanthroline-ATP": a labeling reagent for the active site of ATPases. 21 Aug 47

In order to investigate the roles of the physical states of phospholipid and protein in the enzymatic behavior of the Ca2+ -ATPase from sarcoplasmic reticulum, we have modified the lipid phase of the enzyme, observed the effects on the enzymatic activity at low temperatures, and correlated these effects with spectroscopic measurements of the rotational motions of both the lipid and protein components. Replacement of the native lipids with dipalmitoyl phosphatidylcholine inhibits ATPase activity and decreases both lipid fluidity, as monitored by EPR spectroscopy on a stearic acid spin label, and protein rotational mobility, as monitored by saturation transfer EPR spectroscopy on the covalently spin-labeled enzyme. Solubilization of the lipid-replaced enzyme with Triton X-100 reverses all three of these effects. Ten millimolar CaCl2 added either to the enzyme associated with the endogenous lipids or to the Triton X-100 soulbilized enzyme inhibits both ATPase activity and protein rotational mobility but has no detectable effect on the lipid mobility. These results are consistent with the proposal that both lipid fluidity and protein rotational mobility are essential for enzymatic activity.
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PMID:Effect of the lipid environment on protein motion and enzymatic activity of sarcoplasmic reticulum calcium ATPase. 21 Nov 34

The fate of vanadate (+5 oxidation state of vanadium) taken up by the red cell was studied using EPR spectroscopy. The appearance of an EPR signal indicated that most of the cytoplasmic vanadate is reduced to the +4 oxidation state with axial symmetry characteristic of vanadyl ions. The signal at 23 degrees C was characteristic of an immobilized system indicating that the vanadyl ions in the cytoplasm are associated with a large molecule. [48V]Vanadium eluted with hemoglobin when the lysate from Na3[48V[O4-treated red cells was passed through a Sephadex G-100 column and rabbit anti-human hemoglobin serum caused a hemoglobin-specific precipitation of 48V when added to the red cell lysate. Both results indicate that hemoglobin is the protein which binds cytoplasmic vanadyl ions. However, neither sodium vanadate nor vanadyl sulfate bound to purified hemoglobin in vitro. Finally, transient kinetics of vanadyl sulfate interaction with the sodium-and potassium-stimulated adenosine triphosphatase showed that the +4 oxidation state of vanadium is less effective than the +5 oxidation state in inhibiting this enzyme. These results indicate that oxidation-reduction reactions in the cytoplasm are capable of relieving vanadate inhibition of cation transport.
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PMID:The fate of cytoplasmic vanadium. Implications on (NA,K)-ATPase inhibition. 21 70

Thermoplasma acidophilum, a mycoplasma-like organism, grows optimally at 56 degrees C and pH2. The low temperature extreme of growth is 37 degrees C. The plasma membrane of cells grown at 37 degrees C was isolated and characterized physicobiochemically. Membrane lipids which comprise 25% of the membrane dry weight consist mainly of two repetitively methyl-branched C40 side chains that were ether-linked to two glycerol molecules. The lipid structures were elucidated by combined gas chromatography-mass spectroscopy, direct probe mass spectroscopy and 13C NMR. 37 degrees C-grown cells contained lipids with 42% more pentane cyclization than the 56 degrees C-grown cells. In 37 degrees C-grown cells, phospholipid and serine content decreased by about 10% each, carbohydrate content increased by 5%. EPR studies demonstrated an increase in membrane lipid fluidity of 37 degrees C-grown cells with an upper transition temperature at 35 degrees C which was shifted down by 10 degrees C compared with cells grown at 56 degrees C. Membrane-bound ATPase activities also indicated similar changes upon adaptation. There is a close correlation between membrane fluidity and physiological functioning of this membrane-bound enzyme.
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PMID:Structure of membrane lipids and physico-biochemical properties of the plasma membrane from Thermoplasma acidophilum, adapted to growth at 37 degrees C. 22 Oct 32

The interactions of gadolinium ion, lithium, and two substrate analogues, beta,gamma-imido-ATP (AMP-PNP) and tridentate CrATP, with the calcium ion transport adenosine triphosphatase (Ca2+-ATPase) of rabbit muscle sarcoplasmic reticulum have been examined by using 7Li+ NMR, water proton NMR, and Gd3+ EPR studies. Steady-state phosphorylation studies indicate that Gd3+ binds to the Ca2+ activator sites on the enzyme with an affinity which is approximately 10 times greater than that of Ca2+. 7Li+, which activates the Ca2+-ATPase in place of K+, has been found to be a suitable nucleus for probing the active sites of monovalent cation-requiring enzymes. 7Li+ nuclear relaxation studies demonstrate that the binding of Gd3+ ion to the two Ca2+ sites on Ca2+-ATPase increases the longitudinal relaxation rate (1/T1) of enzyme-bound Li+. The increase in 1/T1 was not observed in the absence of enzyme, indicating that the ATPase enhances the parmagnetic effect of Gd3+ on 1/T1 of 7Li+. Water proton relaxation studies also show that the ATPase binds Gd3+ at two tight-binding sites. Titrations of Gd3+ solutions with Ca2+-ATPase indicate that the tighter of the two Gd3+-binding sites (site 1) provides a ghigher enhancement of water relaxation than the other, weaker Gd3+ site (site 2) and also indicate that the average of the enhancements at the two sites is 7.4. These data, together with a titration of the ATPase with Gd3+ ion, yield enhancements, epsilonB, of 9.4 at site 1 and 5.4 at site 2. Analysis of the frequency dependence of 1/T1 of water indicates that the electron spin relaxation taus of Gd3+ is unusually long (2 X 10(-9) s) and suggests that the Ca2+-binding sites on the ATPase experience a reduced accessiblity of solvent water. This may indicate that the Ca2+ sites on the Ca2+-ATPase are buried or occluded within a cleft or channel in the enzyme. The analysis of the frequency dependence is also consistent with three exchangeable water protons on Gd3+ at site 1 and two fast exchanging water protons at site 2. Addition of the nonhydrolyzing substrate analogues, AMP-PNP and tridenate CrATP, to the enzyme-Gd3+ complex results in a decrease in the observed enhancement, with little change in the dipolar correlation time for Gd3+, consistent with a substrate-induced decrease in the number of fast-exchanging water protons on enzyme-bound Gd3+. From the effect of Gd3+ on 1/T1 of enzyme-bound Li+, Gd3+-Li+ separations of 7.0 and 9.1 A are calculated. On the assumption of a single Li+ site on the enzyme, these distances set an upper limit on the separation between Ca2+ sites on the enzyme of 16.1 A.
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PMID:Lithium-7 nuclear magnetic resonance, water proton nuclear magnetic resonance, and gadolinium electron paramagnetic resonance studies of the sarcoplasmic reticulum calcium ion transport adenosine triphosphatase. 22 3

Membranes of Mycoplasma hominis cells from cultures progressing from the mid to the end of the logarithmic phase of growth became richer in protein, poorer in phospholipids and cholesterol, heavier in density, and more viscous as determined by EPR. The membrane-bound ATPase activity declined steeply. Electrophoretic analysis failed to show marked changes in membrane protein composition on aging, apart from an increase in the staining intensity of one protein band (Mr approximately 130 000) concomitant with a decrease in the staining intensity of several minor protein bands of high molecular weight. To test for possible changes in the disposition of the various membrane proteins on aging of cultures, a comparison was made of the susceptibility of membrane proteins of intact cells and isolated membranes to trypsinization and lactoperoxidase-mediated iodination. The iodination values and the percent of membrane protein released by trypsinization of intact cells were similar in cells from cultures of different ages, indicating no significant changes in the organization of the proteins on the outer surface. On the other hand, trypsinization and iodination of isolated membranes were found to be most markedly affected by the culture age, indicating significant changes in the organization of the proteins on the inner membrane surface. Thus, the iodination values of isolated membranes decreased by almost two fold, while the percentage of protein released from the membrane by trypsin increased from 28% to 50% during the experimental period. It is suggested that aging in M. hominis cultures is accompanied by a continuous increase in the packing density of the protein molecules on the inner surface of the cell membrane.
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PMID:Characterization of the mycoplasma membrane proteins. VI. Composition and disposition of proteins in membranes from aging Mycoplasma hominis cultures. 125 8

The membranous Na+/K(+)-ATPase from Squalus acanthias has been covalently modified on either Class I or Class II sulphydryl groups using derivatives of 3-(maleimidomethyl)-1-oxyl-2,2,5,5-tetramethylpyrrolidine with substituents of different charge and hydrophobicity attached at the remaining unsubstituted position of the pyrrolidine ring. The substituent groups used were a methyl and a hexyl ester, and di- and tri-methylammonium ethyl esters, as well as the parent underivatized compound. Additionally, another series of maleimide-nitroxides differing (by zero to seven intervening atoms) in the length of the linking group between the maleimide and the pyrrolidine moieties was used. The sites of attachment have been characterized in terms of the rotational mobility and environmental polarity by using conventional and saturation transfer EPR spectroscopy of these spin-labelled reagents. This provides a further sub-classification of the primary Class I and Class II SH-groups on the alpha-subunit of the enzyme, which differ both in their reactivity and influence on the Na+/K(+)-ATPase activity.
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PMID:Conventional and saturation transfer EPR spectroscopy of Na+/K(+)-ATPase modified with different maleimide-nitroxide derivatives. 132 55

Spin-label EPR spectroscopy of shark rectal gland Na,K-ATPase modified at cysteine residues with a variety of maleimide-nitroxide derivatives is used to characterize the different classes of sulphydryl groups. The spin-labelled derivatives vary with respect to charge and lipophilicity, and the chemical reactivity towards modification and inactivation of the Na,K-ATPase is dependent on these properties. Ascorbate is used to reduce the spin-labels in situ, and the kinetics of reduction of the protein-bound spin-labels are found also to depend on the nature of the maleimide-nitroxide derivative. The Na,K-ATPase is labelled either at Class I groups (with retention of enzymatic activity) or at Class II groups (where the enzymatic activity is lost). Although Class I groups are labelled more readily than are Class II groups they are only slightly more susceptible to reduction by ascorbate than the Class II groups, indicating no major difference in environment. The spectral difference observed between immobilized and mobile spin-labels with both Class I and Class II groups labelling is not reflected in widely different reduction kinetics for these two spectral components. Solubilization of the enzyme in an active form does not change the protein structure in terms of increased accessibility of the SH-groups to reduction by ascorbate. The results are discussed in terms of the location of the different SH-groups and the origins of the differences in mobility evident in the EPR spectra of the spin-labelled SH-groups.
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PMID:Analysis of thiol-topography in Na,K-ATPase using labelling with different maleimide nitroxide derivatives. 133 3

The chromium moiety of gamma,beta-bidentate CrATP slowly accepts a ligand from the sarcoplasmic reticulum Ca-ATPase to form an exchange inert coordination complex (k + 1 = 0.083 min-1; k - 2 = 0.003 min-1, 37 degrees C, 100 microM CaCl2). The stability of the Cr3+ coordinate bonds allowed the complex to be isolated by filtration techniques at neutral pH without acid precipitation. We found 4-5 nmol of [gamma-32P]CrATP to bind to 1 mg of sarcoplasmic reticulum protein with the subsequent occlusion of 7-8 nmol of 45Ca2+. At 37 degrees C, the CrATP.ATPase complex could be formed in the absence of Ca2+, although the reaction was 2-3 times slower than in the presence of Ca2+. Inhibition by Pi, by orthovanadate, and by fluorescein 5'-isothiocyanate verified that the bound CrATP was at the catalytic site. The site of CrATP attachment was found to be on the A tryptic fragment, possibly on the A2 subfragment. It was determined that Ca2+ binding to high affinity sites on the enzyme controls the rate by which the Cr3+ moiety accepts the ligand from the enzyme. The rate of change in the EPR spectrum of iodoacetamide spin-labeled ATPase was shown to follow the rate of ligand acceptance, rather than the binding of Ca2+ and substrate per se. This particular change has been attributed to the formation of an activated complex that is immediately precursory to phosphorylation and indicates here that this complex cannot be properly formed until the metal has been chelated by the enzyme. It is concluded that control over metal chelation (Cr3+ here, Mg2+ in the normal mechanism) is one means by which Ca2+ activates the enzyme.
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PMID:Interaction of CrATP with the phosphorylation site of the sarcoplasmic reticulum ATPase. 164 96


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