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)

Myosin was incubated with a large excess of exogenous g1, g2 or g3 in 0.6 M KSCN (or in 4 M LiCl) for 1-2 h at 0-2 degrees C. KSCN (or LiCl) was then removed by dialysis. The composition of g-chains in the resulting myosin was analyzed by SDS-gel electrophoresis. When myosin was incubated with g1, the amount of g1 in myosin increased and the increment was nearly counterbalanced by a decrease in g3, whereas an opposite change was observed on incubation with g3. The amount of g2 was not changed by these treatments. The same ATPase activity as that of control myosin was observed in the presence of Ca2+ or EDTA with the myosins incubated with g1, g2, or g3, but the activity in the presence of Mg2+ was about one-half of the control. The Ca2+ sensitivity of actomyosin containing the treated myosins was slightly higher than that of actomyosin containing the control myosin. Spin-labeled g1 or spin-labeled g3 was incorporated into myosin, but the ESR spectra of two spin labels were not distinguishable. No information could be obtained from the ESR spectra by the addition of Ca2+, Mg2+, nucleotides or actin. Inhibition of ATPase activity was observed when SH groups g1 or g3 in myosin were chemically modified.
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PMID:Incubation of myosin with exogenous small components (g1, g2, or g3) in KSCN or LiCl and properties of g-exchanged myosins. 14 65

Pure complexes of dipalmitoyllecithin (DPL, 16:0) which Ca2+, Mg2+ dependent ATPase from sarcoplasmic reticulum are unusual in retaining significant ATPase activity down to about 30 degrees C, well below the transition temperature of the pure lipid at 41 degrees C. A minimum of about 35 lipid molecules per ATPase is required to maintain maximal ATPase activity, but the complexes are progressively and irreversibly inactivated at lower lipid to protein ratios. Complexes containing more than the minimum lipid requirement show very similar temperature profiles of activity about 30 degrees C over a wide range of lipid to protein ratios, up to 1500:1. Spin-label studies indicate that, at lipid to protein ratios of less than about 30 lipids per ATPase, no DPL phase transition can be detected, but at all higher ratios, a phase transition occurs at about 41 degrees C. In all of these complexes there are breaks in the Arrhenius plots of ATPase activity at 27--32 degrees C and at 37.5--38.5 degrees C. Experiments with perturbing agents, such as cholesterol and benzyl alcohol which have well-defined effects on the DPL phase transition, indicate that these breaks in the Arrhenius plots of ATPase activity cannot be attributed to a depressed and broadened phase transition in the lipids near the protein molecules. These results are interpreted as evidence for a phospholipid annulus of at least 30 lipid molecules with interact directly with the ATPase and cannot undergo a phase transition at 41 degrees C. This structural interaction of the ATPase with the annular DPL molecules has a predominant effect in determining the form of the temperature-activity profiles. However, the perturbation of the DPL phase transition does not extend significantly beyond the annulus since a phase transition which starts at 41 degrees C can be detected as soon as extraannular lipid is present in the complexes. We suggest that it may be a general feature of membrane structure that penetrant membrane proteins interact with their immediate lipid environment so as to cause only a minimal perturbation of the lipid bilayer.
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PMID:Annular lipids determine the ATPase activity of a calcium transport protein complexed with dipalmitoyllecithin. 18 11

The interaction of a nitroxide spin-labeled derivative of ouabain with sheep kidney Na,K-ATPase and the motional behavior of the ouabain spin label-Na,K-ATPase complex have been studied by means of electron paramagnetic resonance (EPR) and saturation-transfer EPR (ST-EPR). Spin-labeled ouabain binds with high affinity to the Na,K-ATPase with concurrent inhibition of ATPase activity. Enzyme preparations retain 0.61 +/- 0.1 mol of bound ouabain spin label per mole of ATP-dependent phosphorylation sites, even after repeated centrifugation and resuspension of the purified ATPase-containing membrane fragments. The conventional EPR spectrum of the ouabain spin label bound to the ATPase consists almost entirely (greater than 99%) of a broad resonance at 0 degrees C, characteristic of a tightly bound spin label which is strongly immobilized by the protein backbone. Saturation-transfer EPR measurements of the spin-labeled ATPase preparations yield effective correlation times for the bound labels significantly longer than 100 microseconds at 0 degrees C. Since the conventional EPR measurements of the ouabain spin-labeled Na,K-ATPase indicated the label was strongly immobilized, these rotational correlation times most likely represent the motion of the protein itself rather than the independent motion of mobile spin probes relative to a slower moving protein. Additional ST-EPR measurements of ouabain spin-labeled Na,K-ATPase (a) cross-linked with glutaraldehyde and (b) crystallized in two-dimensional arrays indicated that the observed rotational correlation times predominantly represented the motion of large Na,K-ATPase-containing membrane fragments, as opposed to the motion of individual monomeric or dimeric polypeptides within the membrane fragment. The results suggest that the binding of spin-labeled ouabain to the ATPase induces the protein to form large aggregates, implying that cardiac glycoside induced enzyme aggregation may play a role in the mechanism of action of the cardiac glycosides in inhibiting the Na,K-ATPase.
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PMID:Effects of ouabain on the rotational dynamics of renal Na,K-ATPase studied by saturation-transfer EPR. 131 Dec

Sphingomyelin liposomes and brain microsomes were oxidized by exposure to hydrogen peroxide and ferrous ion. Lipid peroxidation were measured by the formation of thiobarbituric acid- reactive substances (TBAR). Hydroxyl radical was detected using the spin- trapping technique. Incubation of sphingomyelin liposomes with H2O2-Fe2+ resulted in an increase in the formation of TBAR. Na(+)-K(+)-ATPase activity was markedly inhibited and the SH group content decreased during incubation of microsomes in the presence of H2O2-Fe2+. Sodium ferulate effectively inhibited TBAR formation, protected Na(+)-K(+)-ATPase activity and prevented the oxidative modification of SH groups. Spin-trapping experiments showed that sodium ferulate effectively scavenged the hydroxyl radicals.
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PMID:Effect of hydroxyl radical on Na(+)-K(+)-ATPase activity of the brain microsomal membranes. 133 Mar 32

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

Gd3+ ions were bound to the Ca(2+)-transport site of Ca(2+)-transporting ATPase of the sarcoplasmic reticulum (SR-ATPase) and their effect on the ESR spectrum of spin-probes, which were attached to specific sites on SR-ATPase and embedded in the membranous lipid at various depths from the surface of the membrane, was studied. Spin-labeled reagents, 1-oxyl-2,2-dimethyl-oxazolidine derivatives of maleimidoethyl-keto stearate, collectively abbreviated as MSL(m,n) were mainly used for labeling SR-ATPase. They have Cm- and Cn-hydrocarbon chains, respectively, on both sides of the spin label, of which the Cm-hydrocarbon chain is located distal to the carboxyl group of the keto stearate moiety. Paramagnetic interaction between Gd3+ and a spin probe was detected by measuring the decrease in the intensity of the ESR signal of the probe. Displacement of Gd3+ from the Ca(2+)-transport site by Ca2+, which had been confirmed previously by using fluorescently labeled SR-ATPase (described in the preceding article), led to a significant reversal of the paramagnetic effect of Gd3+ on MSL(12,3) and MSL(10,5) attached to SR-ATPase. On the other hand, the effect of Gd3+ was not reversed by Ca2+ when SR-ATPase labeled with MSL(1,14) or a spin-label specific for the cytoplasmic domain was used. These results led us to conclude that the Ca(2+)-transport site of SR-ATPase is located in the membranous region of the molecule, but that the site is not very far from the surface of the membrane of the sarcoplasmic reticulum.
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PMID:Location of the Ca(2+)-transport site of Ca(2+)-transporting ATPase of the sarcoplasmic reticulum as determined by analysis of paramagnetic interaction between Gd3+ ions bound at the transport site and membrane-embedded nitroxide spin probes. 166 2

The rotational motion of an ouabain spin label with sheep kidney Na,K-ATPase has been measured by electron paramagnetic resonance (EPR) and saturation transfer EPR (ST-EPR) measurements. Spin-labelled ouabain binds with high affinity to the Na,K-ATPase with concurrent inhibition of ATPase activity. Enzyme preparations retain 0.61 +/- 0.1 mol of bound ouabain spin label per ATPase alpha beta dimer. The conventional EPR spectrum of the ouabain spin label bound to the ATPase consists almost entirely (greater than 99%) of a broad resonance which is characteristic of a strongly immobilized spin label. ST-EPR measurements of the spin labelled ATPase preparations yield effective correlation times for the bound labels of 209 +/- 11 microseconds at 0 degree C and 44 +/- 4 microseconds at 20 degrees C. These rotational correlation times most likely represent the motion of the protein itself rather than the independent motion of mobile spin probes relative to a slower moving protein. Additional ST-EPR measurements with glutaraldehyde-crosslinked preparations indicated that the observed rotational correlation times predominantly represented the motion of entire Na,K-ATPase-containing membrane fragments, rather than the motion of individual monomeric or dimeric polypeptides within the membrane fragment. The strong immobilization of the ouabain spin label will make it an effective paramagnetic probe of the extracellular surface of the Na,K-ATPase for a variety of NMR and EPR investigations.
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PMID:Saturation transfer EPR measurements of the rotational motion of a strongly immobilized ouabain spin label on renal Na,K-ATPase. 215 81

Delipidation of beef heart electron transport particles with phospholipase A2 has been examined. When the particles were treated with the lipase and subjected to a low bovine serum albumin wash, ATPase activity was unaffected as was the lipid/protein ratio of the particles. However, energisation by ATP/Mg2+ was abolished. Furthermore, unsaturated but not saturated fatty acids discharged the steady-state ATP-driven membrane potential of control samples. When the phospholipase A2 hydrolysis products were removed, inhibition of energy-linked reactions in the lipid-depleted particles was still observed and was interpreted in terms of non-specific leaks in the vesicle membranes, and 'specific' leaks through impaired H+-ATPase complexes. ATPase activity was less susceptible to delipidation than energisation but was, nevertheless, strongly inhibited at 50 percent lipid depletion. Spin label studies indicated a decrease in the fluidity of particle membranes accompanying delipidation. Moreover, the discontinuity seen in Arrhenius plots of ATPase activity was shifted from 17 degrees C (control) to 22 degrees C at 50 percent phospholipid depletion. The data are consistent with a release of unsaturated fatty acids by phospholipase A2 rendering the transport particles both leakier and the membranes less fluid than controls.
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PMID:Effects of delipidation on proton translocation and ATPase activity in beef heart electron transport particles. 288 57

Spin-labeled derivatives of AMP-PCP, ATP, and 2'-deoxy-ATP, with a nitroxide moiety attached to the ribose ring [3'-O-(1-oxy-2,2,5,5-tetramethylpyrroline-3-carbonyl)nucleotide], are used to study the nucleotide binding site stoichiometry of sarcoplasmic reticulum (SR) ATPase. With all derivatives, a maximal binding of 4.5 nmol/mg of SR protein is found, a value close to the number of phosphorylation sites obtained with ATP. The spin-labeled nucleotides cannot be utilized by the enzyme as substrates. Binding of spin-labeled nucleotides is inhibited by labeling the ATPase with fluorescein 5'-isothiocyanate, indicating that all the labeled nucleotide is located at the catalytic site. Additions of spin-labeled ATP to vesicle suspensions during steady turnover demonstrate competitive inhibition of both catalysis and the regulatory effect normally exhibited by ATP. As secondary binding of spin-labeled ATP is not detected at pertinent concentrations, it is suggested that both functions of ATP may be effected through a single site.
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PMID:Interaction of spin-labeled nucleotides with sarcoplasmic reticulum adenosinetriphosphatase. 297 48

Cesium-133 NMR studies have been carried out on suspended human erythrocytes and on perfused rat hearts in media containing CsCl. The resulting spectra exhibit two sharp resonances, arising from intra- and extracellular Cs+, separated in chemical shift by 1.0-1.4 ppm. Thus, intra- and extracellular resonances are easily resolved without the addition of paramagnetic shift reagents required to resolve resonances of the other alkali metal ions. Spin-lattice relaxation times in all cases are monoexponential and significantly shorter (3-4 times) for the intracellular component. When corrections are made for the pulse repetition rate, the total intensity of the intracellular and extracellular Cs+ resonances in erythrocytes is conserved, implying total observability of the intracellular pool. The uptake of Cs+ by erythrocytes occurs at approximately one-third the reported rate for K+ and was reduced by a factor of 2 upon addition of ouabain to the sample. These results indicate that 133Cs NMR is a promising tool for studying the distribution and transport of cesium ions in biological systems and, in some cases such as uptake by cellular Na,K-ATPase, for analysis of K+ ion metabolism.
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PMID:Uptake of cesium ions by human erythrocytes and perfused rat heart: a cesium-133 NMR study. 340 10


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