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Query: UNIPROT:O14944 (EPR)
 13,097 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

The state of existence of cytoplasmic vanadium ion is known to be important: vanadyl ion forms complexes with ATP and vanadate form inhibits (Na++K+)-ATPase. Therefore, the formation of complexes between ATP and vanadyl ion was investigated. Formations of three types of complex were observed: a blue 1:1 complex formed in acidic pH region; a blue 1:1 complex formed in neutral pH region; a green 2:1 complex formed in alkaline pH region. On the basis of the results on potentiometric titration, optical and EPR spectra and 31P- and 13C-NMR spectra, three characteristic types of coordination environment are proposed.
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PMID:Structures of ATP(adenosine triphosphate)-vanadyl complexes. 630 4

Psychotropic drugs, especially neuroleptics, reversibly and noncompetitively inhibit the activity of Na, K-ATPase of the brain and Ca, Mg-ATPase of the sarcoplasmatic reticulum. The inhibitory effect is more pronounced versus the sarcoplasmatic reticulum and less marked versus purified enzymatic preparations. It is not much dependable on variations in the protein concentration of enzymatic preparations and is not related to drug binding to sulfhydryl groups of an enzyme. The inhibitory action of the drugs declines or completely disappears after treating the membranous preparations with phospholipase A. That psychotropic drugs have a predominant effect on the lipid structure of the membrane was supported during examination of EPR spectra of lipid spin labels.
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PMID:[Mechanisms of the action of psychotropic preparations on transport ATPases]. 645 55

A spin-labeled derivative of porcine gastric H/K-ATPase with high ATP hydrolyzing activity (77 mumol of Pi/(mg.h)) has been prepared. Over 65% of initial ATPase activity (115 mumol of Pi/(mg.h)) was preserved after complete reaction of the enzyme with the lysine reactive nitroxide spin-labeled TEMPO isothiocyanate (TITC). In contrast, rapid and complete loss of ATPase activity occurred after reaction of the enzyme with the lysine directed fluorescent probe FITC. Conventional EPR spectra of TITC labeled H/K-ATPase reflected mainly the slow rotational diffusion of the enzyme in the membrane. An upper limit enzyme intramembranous radius of 108 A was calculated on the basis of rotational correlation times estimated from saturation transfer (ST) EPR spectral lineshapes. Conventional EPR spectra exhibited two major components corresponding to at least two populations of strongly constrained spin-labels. Difference spectroscopy revealed that the proportion of these two components changed markedly with temperature. Moreover, the proportion of the components was sensitive to the presence of the activating ionic ligands Mg2+ and ATP, which induce enzyme conformational transitions, and to the reversible inhibitor SCH 28080, which binds to the K+ sensitive form of the enzyme. These findings show that EPR spectroscopy is able to report functionally coupled conformational changes of gastric H/K-ATPase and imply that the spin-labels are attached to lysines within functionally important regions of the enzyme.
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PMID:The conformations of a functional spin-labeled derivative of gastric H/K-ATPase investigated by EPR spectroscopy. 777 84

We have identified the most probable protein ligands at the catalytic M3 and noncatalytic M2 metal-binding sites in the spinach chloroplast F1-ATPase (CF1) and here propose possible residues in the protein sequence for these ligands in latent CF1 in the absence of nucleotide. The changes in the metal ligands at these sites upon binding of nucleotide to the N2 and N3 sites and upon activation of latent CF1 provide a possible molecular basis for inhibition of ATPase activity by free metal, for the lack of activity in the latent state, and for the gating mechanism of the ATPase H+ pump. To these ends, the Mg2+ analogue, vanadyl (VIV = O)2+, was used as a paramagnetic probe at the M2 and M3 metal-binding sites. EPR and ESEEM spectra of VO2+ were obtained, and simulations of the full EPR spectra imply the ligand sets at the different metal-binding sites. When VO2+ is added to CF1 in the absence of ATP, the most likely set of ligands at the M2 site are 1 ROH (alpha T176), 2 H2O, and 1 RCOO- (alpha D269 or alpha D270), where the suggested amino acid designations of the residues are given in parentheses according to the mitochondrial sequence. Evidence suggests a possible axial nitrogen ligand at this site (alpha K175). When the M2 site is filled by addition of VO2+ and ATP, the metal binds as a second species in which N2-bound ATP and M2-bound VO2+ form a monodentate complex with concomitant exchange of the equatorial protein ligands by 3 H2O.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Effects of nucleotides on the protein ligands to metals at the M2 and M3 metal-binding sites of the spinach chloroplast F1-ATPase. 788 Aug 23

We have used saturation transfer electron paramagnetic resonance (ST-EPR) to measure the microsecond rotational motion of actin-bound myosin heads in spin-labeled myofibrils in the presence of the ATP analogs AMPPNP (5'-adenylylimido-diphosphate) and ATP gamma S (adenosine-5'-O-(3-thiotriphosphate)). AMPPNP and ATP gamma S are believed to trap myosin in two major conformational intermediates of the actomyosin ATPase cycle, respectively known as the weakly bound and strongly bound states. Previous ST-EPR experiments with solutions of acto-S1 have demonstrated that actin-bound myosin heads are rotationally mobile on the microsecond time scale in the presence of ATP gamma S, but not in the presence of AMPPNP. However, it is not clear that results obtained with acto-S1 in solution can be extended to actomyosin constrained within the myofibrillar lattice. Therefore, ST-EPR spectra of spin-labeled myofibrils were analyzed explicitly in terms of the actin-bound component of myosin heads in the presence of AMPPNP and ATP gamma S. The fraction of actin-attached myosin heads was determined biochemically in the spin-labeled myofibrils, using the proteolytic rates actomyosin binding assay. At physiological ionic strength (mu = 165 mM), actin-bound myosin heads were found to be rotationally mobile on the microsecond time scale (tau r = 24 +/- 8 microseconds) in the presence of ATP gamma S, but not AMPPNP. Similar results were obtained at low ionic strength, confirming the acto-S1 solution studies. The microsecond rotational motions of actin-attached myosin heads in the presence of ATP gamma S are similar to those observed for spin-labeled myosin heads during the steady-state cycling of the actomyosin ATPase, both in solution and in an active isometric muscle fiber. These results indicate that weakly bound myosin heads, in the pre-force phase of the ATPase cycle, are rotationally mobile, while strongly bound heads, in the force-generating phase, are rotationally immobile. We propose that force generation involves a transition from a dynamically disordered crossbridge to a rigid and stereospecific one.
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PMID:Rotational dynamics of actin-bound intermediates of the myosin adenosine triphosphatase cycle in myofibrils. 791 93

Conventional EPR studies of muscle fibers labeled with a novel alpha-iodoketo spin label at Cys-707 of the myosin head revealed substantial internal domain reorganization on the addition of ADP to rigor fibers. The spin probes that are well-ordered in the rigor state become disordered and form two distinct populations. These orientational changes do not correspond to rotation of the myosin catalytic domain as a whole because other probes (maleimide and iodoacetamide nitroxides attached to the same Cys-707 of myosin head) report only a small (5-10 degrees) torsional rotation and little or no change in the tilt angle [Ajtai et al. (1992) Biochemistry 31, 207-17; Fajer (1994) Biophys. J. 66, 2039-50]. In the presence of ADP, the labeled domain becomes more flexible and executes large-amplitude microsecond motions, as measured by saturation-transfer EPR with rates (tau r = 150 microseconds) intermediate between the rotations of detached (tau r = 7 microseconds) and rigor heads (tau r = 2500 microseconds). This finding contrasts with an absence of global motion of the myosin head in ADP (tau r = 2200 microseconds) as reported by the maleimide spin label. Our results imply that the myosin head in a single chemical state (AM.ADP) is capable of attaining many internal configurations, some of which are dynamic. The presence of these slow structural fluctuations might be related to the slow release of the hydrolysis products of actomyosin ATPase.
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PMID:Myosin catalytic domain flexibility in MgADP. 794 41

Vanadyl (VIV=O)2+ was used as a paramagnetic probe at the M2 and M3 metal-binding sites of the spinach chloroplast F1-ATPase (CF1) in order to detect interaction of the metals with nucleotides. The M2 site can exist in two forms in the presence of ATP. When ATP and VO2+ are added in a 1.5:1 ratio to CF1, the VO2+ EPR spectrum is identical to that of CF1-VO2+ in the absence of ATP. When the M2 site is filled by the addition of ATP and VO2+ in a 3:1 ratio, the VO2+ binds to M2 in a second form with equatorial coordination to a single phosphate. The treatments required to deplete CF1 of the monodentate VO2+(-)nucleotide complex indicate that the VO2+ is coordinated to the ATP at the nonacatalytic N2 site. The presence of uncomplexed nucleotide appears to induce formation of the second form, possibly via ATP binding to the N3 site. This change in coordination at the M2 noncatalytic site may regulate the ATPase activity of CF1. The M3 site also exists in two forms: (i) in latent CF1, no phosphate coordination is evident; and (ii) after the ATPase has been activated, the EPR line shape is consistent with the two phosphates from ADP at N3 coordinated to the VO2+ at M3. This work establishes a connection between the metal- and nucleotide-binding sites as M2-N2 and M3-N3.
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PMID:Coordination of nucleotides to metals at the M2 and M3 metal-binding sites of spinach chloroplast F1-ATPase. 806 Sep 67

We describe here a new procedure permitting rapid (12-13 h) isolation of a pure oxygen-evolving photosystem II (PSII) core complex from the cyanobacterium Synechocystis PCC 6803. This procedure involves dodecyl maltoside extraction of thylakoid membranes followed by single-step column chromatography using a weak anion-exchanger. SDS-PAGE and immunoblotting show that the complex consists of five intrinsic membrane proteins (CP47, CP43, D1, D1, and cyt b559), one extrinsic protein (MSP), and one unknown protein with a molecular mass of approximately 26 kDa. A chemical and functional analysis, normalized to 2 molecules of pheophytin a, indicates that this PSII core complex contains 1 photoactive plastoquinone, QA, 4 manganese atoms, 38 chlorophyll a molecules, 1 cytochrome b559, 2 plastoquinone-9, and 9-10 beta-carotenes. The complex exhibits high rates of oxygen evolution, typically 2400-2600 mumol of O2 (mg of Chl)-1 h-1 in the presence of 2,5-dichlorobenzoquinone as an artificial electron acceptor with a pH optimum of 6.5. A strong light minus dark multiline EPR signal, arising from the S2 state of the oxygen-evolving complex (OEC), is observed at 10 K following illumination at 198 K. The determination of the absolute oxygen yield per saturating microsecond flash indicates that essentially all of the PSII centers contain functional oxygen-evolving complexes. This point is further supported by the absence of photoaccumulation, upon room temperature illumination, of the immediate oxidant of the OEC, redox-active tyrosine, YZ.. On the basis of EPR spectra, oxidized minus reduced difference spectra, and SDS-PAGE, the preparation contains on a per mole basis with PSII only trace amounts of PSI (approximately 0.04), cytochrome b6/f complex (< or = 0.01), and ATPase (< or = 0.05). All of these results indicate that this PSII preparation is to date the most highly purified oxygen-evolving core complex from Synechocystis 6803 that retains all of the reaction centers active for oxygen evolution. As Synechocystis 6803 is being used extensively for site-directed mutagenesis of PSII, this preparation is particularly valuable for spectroscopic and biochemical analyses of PSII from wild-type and from site-directed mutants.
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PMID:Biochemical and spectroscopic characterization of a new oxygen-evolving photosystem II core complex from the cyanobacterium Synechocystis PCC 6803. 816 15

Vanadyl, (V = O)2+, is able to substitute for Mg2+ as a cofactor for ATPase activity catalyzed by the chloroplast F1-ATPase (CF1). Mg2+-dependent ATPase activity was also observed with CF1 that contained VO(2+)-ATP bound specifically to the noncatalytic N2 site. Modulation of the Mg(2+)-ATPase activity induced by VO2+ bound at this site indicates that the metal bound to the noncatalytic site affects catalytic activity. When CF1 is depleted of nucleotides from all but the N1 site, a single Mg2+ remains bound at a site designated M1. Addition of VO2+ to the depleted protein gives rise to an EPR spectrum characteristic of a CF1-bound VO2+ species. The binding curve of the VO2+ complex to latent, nucleotide-depleted CF1 was determined by the integrated intensities of the -5/2 parallel peak in the EPR spectrum as calibrated using atomic absorption spectroscopy. Under these conditions, VO2+ binds cooperatively to approximately two sites designated M2 and M3. Three-pulse ESEEM spectra of the CF1-VO2+ complex contain two intense modulations with frequencies and field-dependent behavior that show that they are from a directly coordinated 14N nucleus. Analysis of the bound VO2+ by ENDOR spectroscopy revealed the presence of a single group of protons associated with an equatorial amino or water ligand that is exchangeable with solvent. Using the additivity relation for hyperfine coupling, the most probable set of equatorial ligands to the VO2+ bound to CF1 under these conditions consists of one lysine nitrogen, two carboxyl oxygens from aspartate or glutamate, and one water.
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PMID:Characterization of ligands of a high-affinity metal-binding site in the latent chloroplast F1-ATPase by EPR spectroscopy of bound VO2+. 816 51

Localization of the PCMB-R spin label and benzocarboline probe bound with the purified preparation of pig kidney-Na+, K(+)-ATPase relative to active site of the enzyme was studied by EPR method. The number of Mn2+ ions in active site of the enzyme as well as that bound with lipids was determined from EPR spectra of paramagnetic manganese ions replacing magnesium ions were measured in frozen protein samples of Na2+, K(+)-ATPase at 77 K. It has been found that sulfhydryl group of the enzyme modified by PCMB-R and benzocarboline probe are placed at distances 38 A and 50 A, respectively, from Mn2+ ions in the active site of Na+, K(+)-ATPase. Evaluation of the immersion depth of the nitroxyl radical into protein globule showed that benzocarboline probe was immobilized near the macromolecular protein surface; there are two bound probe sites, distinguished by accessibility of ferricyanide ions.
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PMID:[Interaction of spin labels and probes with Na+,K+-ATPase]. 829 Nov 40


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