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)

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

We have used saturation-transfer electron paramagnetic resonance (ST-EPR) to measure the submillisecond rotational motions of actin-bound myosin heads in active myofibrils. The cross-bridges were spin-labeled with a maleimide nitroxide derivative (MSL) that has previously been shown to undergo microsecond rotational motions on actin-bound myosin heads in solution during steady-state ATPase activity at low ionic strength [Berger, C. L., Svensson, E. C., & Thomas, D. D. (1989) Proc. Natl. Acad. Sci. U.S.A. 86, 8573]. To determine whether this is also true for cross-bridges in the myofibrillar lattice under physiological buffer conditions, we have performed ST-EPR experiments during the brief steady state following photolysis of caged ATP in a suspension of spin-labeled myofibrils. The myofibrils were partially cross-linked with EDC [1-ethyl-3-[3-(dimethylamino)propyl]carbodiimide] to prevent their shortening upon activation. The fraction of actin-attached myosin heads was determined biochemically at physiological ionic strength in the active myofibrils, using the proteolytic rates acto-myosin binding assay [Duong, A. M., & Reisler, E. (1989) Biochemistry 28, 3502]. These data were then used to correct the ST-EPR spectra of active myofibrils for the presence of unattached myosin heads, which were assumed to undergo the same motions as in relaxation. At physiological ionic strength (mu = 165 mM), actin-bound myosin heads were found to have considerable microsecond rotational motion (tau r = 3.5 +/- 1.1 microseconds) in the active myofibrils. Similar results (tau r = 3.2 +/- 0.8 microseconds) were obtained with active myofibrils at low ionic strength (mu = 45 mM), confirming the work done in solution. Thus, under physiological conditions and even within the constraints of the myofibrillar lattice, actively cycling actin-attached myosin heads are rotationally mobile on the microsecond time scale. Since partially EDC-fixed myofibrils are an excellent analog of isometrically contracting muscle fibers in solution, it is likely that these microsecond rotational motions are directly related to the molecular mechanism of muscle contraction in vivo.
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PMID:Rotational dynamics of actin-bound myosin heads in active myofibrils. 838 91

We have used time-resolved electron paramagnetic resonance spectroscopy and caged ATP to detect nucleotide-induced changes in the conformational state of spin-labeled myosin heads (IASL-S1). Changes in the internal rotational dynamics of IASL-S1 were monitored with millisecond time resolution during the pre-steady-state phase of ATP hydrolysis. The changes in the internal protein dynamics were rigorously correlated with specific biochemical kinetic transitions, allowing us to observe directly the dynamic sequence of structural changes in IASL-S1 during the binding and hydrolysis of ATP. When caged ATP was photolyzed (producing 500 microM ATP) in the presence of 100 microM IASL-S1, the EPR signal intensity rose transiently to the steady-state ATPase level, indicating increased rotational motion about the SH1 region of the myosin head. Kinetic and spectral analyses have resolved two phases of this transient, one representing the population of the M*.ATP state and the other representing the population of the M**.ADP.Pi state. We conclude that two motionally distinct states of the myosin head are present during ATP hydrolysis and that these states represent distinct conformational states that can be correlated with specific biochemical intermediates. Since specific labeling of myosin heads with IASL has been achieved in skinned muscle fibers, this study establishes the feasibility for the first direct detection and resolution of myosin's conformational transients during muscle contraction.
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PMID:Transient detection of spin-labeled myosin subfragment 1 conformational states during ATP hydrolysis. 839 68

We have studied the molecular mechanism of Ca-ATPase activation in sarcoplasmic reticulum (SR) by the volatile anesthetic halothane. Using time-resolved phosphorescence anisotropy, we determined the rotational correlation times and mole fractions of different oligomeric states of the enzyme, as a function of halothane and temperature. Lipid fluidity was measured independently, using EPR of spin-labeled lipids. At 4 and 7 degrees C, the principal effects of halothane were to increase the activity of the Ca-ATPase and to promote the formation of monomers and dimers of the enzyme from larger aggregates. At higher temperatures (up to 25 degrees C), halothane activated the enzyme, but to a lesser extent than observed at lower temperatures. While the functional effects of halothane were temperature dependent, the effects of halothane on lipid fluidity and protein aggregation state were similar at all temperatures tested. We conclude that at low temperatures Ca-ATPase activity is dominated by aggregation state, so halothane activates the enzyme primarily by promoting the formation of monomers and dimers of the enzyme from larger aggregates. At higher temperatures, the activity of the enzyme is dominated by lipid fluidity, so halothane activates the enzyme by increasing the lipid fluidity. The physical mechanism of Ca-ATPase activation, dominated by aggregation state at low temperature and lipid fluidity at higher temperature, provides an explanation for the break in the Arrhenius plot of Ca-ATPase activity (in the absence of halothane) at approximately 20 degrees C.
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PMID:Molecular mechanism of Ca-ATPase activation by halothane in sarcoplasmic reticulum. 839 42

We have used a recently synthesized indane-dione spin label (2-[-oxyl-2,2,5,5-tetramethyl-3-pyrrolin-3-yl)methenyl]in dane-1,3-dione (InVSL) to study the rotational dynamics of myosin, with saturation-transfer electron paramagnetic resonance (ST-EPR). To determine effective rotational correlation times (tau effr) from InVSL spectra, reference spectra corresponding to known correlation times (tau r) were obtained from InVSL-hemoglobin undergoing isotropic rotational motion in aqueous glycerol solutions. These spectra were used to generate plots of spectral parameters vs. tau r. These plots should be used to analyze ST-EPR spectra of InVSL bound to other proteins, because the spectra are different from those of tempo-maleimide-spin-labeled hemoglobin, which have been used previously as ST-EPR standards. InVSL was covalently attached to the head (subfragment-1; S1) of myosin. EPR spectra and K/EDTA-ATPase activity showed that 70-95% of the heads were labeled, with > or = 90% of the label bound to either cys 707 (SH1) or cys 697 (SH2). ST-EPR spectra of InVSL-S1 attached to glass beads, bound to actin in myofibrils, or precipitated with ammonium sulfate indicated no submillisecond rotational motion. Therefore, InVSL is rigidly immobilized on the protein so that it reports the global rotation of the myosin head. The ST-EPR spectra of InVSL-myosin monomers and filaments indicated tau effr values of 4 and 13 microseconds, respectively, showing that myosin heads undergo microsecond segmental rotations that are more restricted in filaments than in monomers. The observed tau effr values are longer than those previously obtained with other spin labels bound to myosin heads, probably because InVSL binds more rigidly to the protein and/or with a different orientation. Further EPR studies of InVSL-myosin in solution and in muscle fibers should prove complementary to previous work with other labels.
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PMID:Saturation transfer electron parametric resonance of an indane-dione spin-label. Calibration with hemoglobin and application to myosin rotational dynamics. 839 49

Alkanols and tertiary amine derivative local anesthetics modify the activity of Ca(2+)-ATPase. In order to investigate the primary binding sites, associated to the functional changes, sarcoplasmic reticulum (SR) Ca(2+)-ATPase was labeled with maleimide derivative spin labels which bind covalently to SH groups of cysteine residues and allow to probe the regions of the protein close to those residues. The EPR measurements showed motional constraints induced by drug-treatment which indicate changes in the enzyme dynamics and structure. n-Alkanols are shown to affect some of the protein-bound labels by restricting their motion. There is, however, no correlation between the functional effects and the observed motional restriction, in the sense that concentrations of the different alcohols leading to the same functional effects do not induce the same degree of restriction. Dibucaine and tetracaine at functional relevant concentrations also restrict the movement of protein bound labels. But, in this case, correlation between spectral changes and functional effects is observed.
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PMID:Interaction of alkanols and local anesthetics with spin-labeled Ca(2+)-ATPase of sarcoplasmic reticulum vesicles. 866 13

The metal binding sites of isolated F1 ATPase from spinach chloroplasts (CF1) and from the thermophilic bacterium Bacillus PS3 (TF1) have been studied by EPR and pulsed EPR spectroscopy using Mn(II) as a paramagnetic probe. After dialysis in the presence of EDTA, purified CF1 retains 0.14 +/- 0.07 Mg(II) and approximately 0.75 +/- 0.25 ADP. TF1 retains 0.31 +/- 0.03 Mg(II) and 0.08 +/- 0.01 nucleotide (ADP + ATP) after the same treatment. Supplementing known quantities of Mn(II) to metal-depleted CF1 allowed a spectroscopic characterization of the bound Mn(II) cations, for which the EPR spectra at X- and Q-band are reported. The zero field splitting parameters of Mn(II) are derived from the simulation of the EPR signal recorded at Q-band for a sample supplemented with 0.3 Mn/CF1. The values, magnitude of D approximately 200 x 10(-4) cm-1 and magnitude of E approximately 40 x 10(-4) cm-1 suggest that the Mn(II) binds to CF1 in a slightly distorted environment. The ESEEM spectra of complexes of Mn(II) with CF1 were also recorded for different Mn/CF1 ratios. For a complex with 0.8 Mn/CF1, the ESEEM spectrum shows two frequencies at 3.7 and 8.6 MHz that are attributed to the magnetic coupling with 31P with a hyperfine coupling constant of magnitude of A approximately 5.3 MHz, reflecting the interaction with a phosphate group from the endogenous ADP molecule. This demonstrates close proximity of the strong affinity metal site M1 and the endogenous ADP binding site N1, and binding of the ADP beta-phosphate to the divalent metal cation. For Mn(II) complexes with higher Mn/CF1 ratios, new frequency components below approximately 5 MHz are resolved in the spectra in addition to the peaks from 31P. From a comparison of the CF1 spectra and their magnetic field dependence across the Mn(II) EPR line shape with those of Mn(II) complexes with imidazole, glycine, poly-L-lysine, and nucleotide ligands, it is concluded that additional metal binding sites are filled at higher Mn contents and that these involve 14N donors. It is suggested that the most probable set of ligands of the divalent metal(s) for these additional metal sites in CF1 includes a lysine residue, in line with a previous proposal [Houseman, A. L. P., Morgan, L., LoBrutto, R., & Frasch, W. D. (1994) Biochemistry 33, 4910-4917]. Similar experiments for a Mn(II) complex with TF1 (0.4 Mn/TF1) showed no interaction with 31P; instead modulations are detected in the ESEEM below approximately 5 MHz that are attributed to a 14N ligand. This is tentatively attributed to the deprotonated amine of Lys-162 from a beta subunit, on the basis of the structural data available for the mitochondrial F1 complex. Addition of the substrate ATP to this Mn.TF1 complex leads to the formation of a ternary Mn.TF1.ATP complex with coordination of the Mn(II) by a phosphate group from the ATP as judged from the ESEEM results (magnitude of A(31P) approximately 4.5 MHz). An increase in the hyperfine coupling constant of 31P of the phosphate bound to Mn(II) to magnitude of A(31P) approximately 5.1 MHz is observed after incubation of the ternary complex at room temperature. This is interpreted as a significant rearrangement of the coordination sphere of the Mn(II) in the M1 site of the Mn.TF1.ATP complex and may reflect conformational changes of catalytic significance that occur in the nucleotide binding site during unisite hydrolysis of ATP to ADP by this complex.
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PMID:Metal binding sites of H(+)-ATPase from chloroplast and Bacillus PS3 studied by EPR and pulsed EPR spectroscopy of bound manganese(II). 870 62

We have used spin-label EPR spectroscopy to examine possible alterations in protein-protein interactions that accompany the activation of the cardiac sarcoplasmic reticulum (SR) Ca-ATPase following the phosphorylation of phospholamban (PLB). Using a radioactive derivative of a maleimide spin label (MSL), we have developed conditions for the selective spin-labeling of the Ca-ATPase in both native cardiac and skeletal sarcoplasmic reticulum membranes. The rotational dynamics of the cardiac and skeletal Ca-ATPase isoforms in native SR membranes were measured using saturation transfer EPR. We report that the phosphorylation of PLB in cardiac SR results in a (1.8 +/- 0.2)-fold reduction in the overall rotational mobility of the Ca-ATPase. The alteration in the rotational dynamics of the Ca-ATPase is the direct result of the phosphorylation of PLB, and is not related to the phosphorylation of the Ca-ATPase or any other SR proteins since no alteration in the ST-EPR spectrum is observed as a result of conditions that phosphorylate the cardiac Ca-ATPase with ATP. Neither do the use of conditions that activate the Ca-ATPase in cardiac SR result in the alteration of the rotational dynamics or catalytic properties of the Ca-ATPase in skeletal SR where PLB is not expressed. Measurements of the rotational dynamics of stearic acid spin labels (SASL) incorporated into cardiac SR membranes with a nitroxide at the 5- and 12-positions using conventional EPR indicate that there is virtually no difference in the lipid acyl chain dynamics in cardiac SR membranes upon the phosphorylation of PLB. These results indicate that the decrease in the rotational dynamics of the Ca-ATPase in cardiac SR membranes associated with the phosphorylation of PLB is related to enhanced interactions between individual Ca-ATPase polypeptide chains due to (i) an alteration in the spatial arrangement of cardiac Ca-ATPase polypeptide chains within a defined oligomeric state or (ii) increased protein-protein associations. We suggest that altered interactions between Ca-ATPase polypeptide chains and PLB serves to modulate the activation barrier associated with calcium activation of the Ca-ATPase in cardiac SR membranes.
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PMID:Phosphorylation of phospholamban by cAMP-dependent protein kinase enhances interactions between Ca-ATPase polypeptide chains in cardiac sarcoplasmic reticulum membranes. 878 78

The mutation E204Q in the beta subunit of the chloroplast F1-ATPase was made by biolistic transformation of Chlamydomonas reinhardtii. The yield of chloroplast F1-ATPase (CF1) purified from thylakoids was unaltered, suggesting that the mutation did not affect protein assembly. However, photoautotrophic growth of Chlamydomonas strains containing beta E204Q was virtually abolished, and the effect of the mutation on the light-driven ATPsynthase activity catalyzed by purified thylakoids was comparable to the change in the photoautotrophic growth rate. The loss of ATPsynthase activity in the mutant was not the result of uncoupling. Addition of wild-type CF1 to mutant thylakoids depleted of CF1 reconstituted ATPsynthase activity indicating that the mutation did not affect assembly of F0. Furthermore, the mutant CF1F0 was capable of catalyzing ATPase-dependent proton pumping as measured by fluorescence quenching of 9-amino acridine. Although the mutation significantly affected the apparent kcat/K(m) of the Mg(2+)-ATPase activity of the purified CF1-ATPase, no significant effect on the apparent kcat was observed with the mutant compared to wild-type. No significant changes in the ability of Mg2+ or Mn2+ to serve either as a cofactor or as an inhibitor of ATPase activity were observed in the mutants relative to the wild-type CF1-ATPase. EPR spectra were also taken of VO2+ bound at catalytic site 3 in its latent form. In a large fraction of the latent enzyme, a carboxyl group has displaced the nucleotide-phosphate coordination to the metal which results in the free-metal inhibited form (M3). No significant effects on the gII and AII 51V hyperfine parameters were observed between wild-type and mutant. However, the mutation increased the abundance of the M3 form relative to the M3-N3 form (metal-nucleotide-coordinated form). On the basis of these results, beta E204 is not the carboxyl group that displaces the nucleotide phosphate as a ligand to form the free-metal inhibited enzyme form which predominates in site 3 in the latent state. Instead, the data are consistent with a role in which beta E204 is essential to protonate an inorganic phosphate-oxygen to make that oxygen a good leaving group to facilitate ATP synthesis and, via this role in H-bonding, increases the abundance of the functional metal-nucleotide complex bound to the catalytic site.
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PMID:Catalytic and EPR studies of the beta E204Q mutant of the chloroplast F1-ATPase from Chlamydomonas reinhardtii. 881 Sep 28

Lipid peroxidation is considered as one of the manifestations of cellular damage in the toxicity of ochratoxin A (OA). OA; its three natural analogs, OB, OC, and Oalpha; and four synthetic analogs, d-OA, the ethylamide of OA (OE-OA), O-methylated OA (OM-OA), and the lactone-opened OA (OP-OA) were used to study free radical generation in bacteria with Bacillus brevis as a model system. The uptake of the different ochratoxins by B. brevis varied substantially depending on the molecular structures. Electron paramagnetic resonance spectroscopy using alpha-(4-pyridyl-1-oxide)-N-tert-butyl nitrone as a spin trapping agent showed an enhanced free radical generation due to the addition of OA and most of the analogs. The EPR signals could be further enhanced by the addition of Ca2+, a calcium ionophore and an ATPase uncoupler, whereas they were eliminated by incubating the growing cells with vitamin E. The spin adduct hyperfine splitting constants indicate the presence of alpha-hydroxyethyl radicals resulting from generated hydroxyl radicals, which are trapped by alpha-(4-pyridyl-1-oxide)-N-tert-butyl nitrone. The results further suggest that OA induces free radical production in this model system by enhancing the permeability of the cellular membrane to Ca2+.
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PMID:Free radical generation as induced by ochratoxin A and its analogs in bacteria (Bacillus brevis). 891 Mar 17


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