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1.
Lithium-7 nuclear magnetic resonance studies are used to characterize the binding of monovalent cations and substrate analogs to the (Na+ + K+)-ATPase. Li+ substitutes for K+ in the activation of the ATPase, while the longitudinal relaxation rate, , of 7Li+ is increased upon binding of either Mn2+ or CrATP to the enzyme. The effects of Mn2+ are consistent with the existence of a Li+ binding site 7.2A from the single catalytically active Mn2+ site on the ATPase. Temperature effects on the observed relaxation rates indicate that exchange of Li+ at the observed site is rapid, while the effects of added Na+ and K+ suggest that the observed site is a K+-type site not previously observed by other methods. These experiments also demonstrate that Li+ should be superior to other nuclei as NMR probes of the (Na+ + K+)-ATPase. 相似文献
2.
Characterization of ATP binding sites of sheep kidney medulla (Na+ + K+)--ATPase using CrATP 总被引:1,自引:0,他引:1
C M Grisham 《Journal of inorganic biochemistry》1981,14(1):45-57
The nucleotide substrate sites of sheep kidney medulla (NA+ + K+)-ATPase are characterized using CrATP, a paramagnetic, substitution-inert substrate analogue probe. The paramagnetic effect of CrATP on 1/T1 of water protons of water protons is enhanced upon complexation with the enzyme. Titrations of the enzyme with CrATP in the presence of Na+ and K+ yielded characteristic enhancements for the binary enzyme-CrATP and ternary enzyme-Mg2+-CrATP complexes of 3.3 and 3.6 and dissociation constants for CrATP of 5 and 12 microM, respectively. Substitution of Li+ for K+ in these titrations did not substantially alter the titration behavior. From the frequency dependence of 1/T1, the correlation time, tau c, for the dipolar water proton-CrATP interaction is 2.7 x 10(-10) sec, indicating that tau c is dominated by tau s, the electron spin relaxation time of Cr3+. The paramagnetic effect of enzyme-bound Mn2+ on 1/T1 of water protons decreases upon the addition of CrATP. Titration of the binary enzyme-Mn2+ complex with CrATP decreases the characteristic enhancement due to Mn2+ from 6.6-8.0 to 1.5. The failure to observe free Mn2+ epr signals in solutions of the ATPase, Mn2+, and CrATP demonstrate that this decrease in epsilon Mn is due to cross-relaxation between Mn2+ and Cr3+ bound simultaneously to the enzyme, and not to displacement of Mn2+ from the enzyme by CrATP. The relaxation rate, 1/T1, of 7Li+ is increased upon addition of CrATP to solutions of the ATPase, indicating that the sites for Li+ and CrATP are close on the enzyme. A Cr3+-Li+ distance of 4.8 +/- 0.5 angstrom is calculated from that data. 相似文献
3.
The sensitivity of the (Na+ + K+)-ATPase in human red cell membranes to inhibition by Ca2+ is markedly increased by the addition of diluted cytoplasm from hemolyzed human red blood cells. The concentration of Ca2+ causing 50% inhibition of the (Na+ + K+)-ATPase is shifted from greater than 50 microM free Ca2+ in the absence of hemolysate to less than 10 microM free Ca2+ when hemolysate diluted 1:60 compared to in vivo concentrations is added to the assay mixture. Boiling the hemolysate destroys its ability to increase the sensitivity of the (Na+ + K+)-ATPase to Ca2+. Proteins extracted from the membrane in the presence of EDTA and concentrated on an Amicon PM 30 membrane increased the sensitivity of the (Na+ + K+)-ATPase to Ca2+ in a dose-dependent fashion, causing over 80% inhibition of the (Na+ + K+)-ATPase at 10 microM free Ca2+ at the highest concentration of the extract tested. The active factor in this membrane extract is Ca2+-dependent, because it had no effect on the (Na+ + K+)-ATPase in the absence of Ca2+. Trypsin digestion prior to the assay destroyed the ability of this protein extract to increase the sensitivity of the (Na+ + K+)-ATPase to Ca2+. 相似文献
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Spinach chloroplasts were able to photophosphorylate the ADP analog α,β-methylene adenosine 5′-diphosphate (AOPCP). Phosphorylation of AOPCP was catalyzed by chloroplasts that were washed or dialyzed to remove free endogenous nucleotides. In the presence of glucose, hexokinase, AOPCP and 32Pi, the 32P label was incorporated into α,β-methylene adenosine 5′-triphosphate (AOPCPOP).In contrast to photophosphorylation of AOPCP, the ATP analog AOPCPOP was a poor substrate for the ATP-Pi exchange reaction and its hydrolysis was neither stimulated by light and dithiothreitol nor inhibited by Dio-9.Photophosphorylation of AOPCP was inhibited by the α,β- and β,γ-substituted methylene analogs of ATP, while phosphorylation of ADP was unaffected by them. The ATP-Pi exchange was also unaffected by both ATP analogs, while the weak AOPCPOP-Pi exchange was inhibited by the β,γ-methylene analog of ATP.Direct interaction of methylene analogs with the chloroplast coupling factor ATPase was indicated by the enzymatic hydrolysis of AOPCPOP on polyacrylamide gels. 相似文献
7.
Andrei V. Astashkin Arnold M. Raitsimring F. Ann. Walker Christopher Rensing Megan M. McEvoy 《Journal of biological inorganic chemistry》2005,10(3):221-230
Electron paramagnetic resonance (EPR) spectroscopy has been used to structurally characterize the copper-binding site in CusF protein from Escherichia coli. The EPR spectra indicate a single type II copper center with parameters typical for nitrogen and oxygen ligands (A~200 G, g~2.186, g~2.051). The pulsed EPR data show that one of the ligands to Cu2+ is an imidazole ring of a histidine residue. The remote amino nitrogen of this imidazole ring is readily observed by electron spin-echo envelope modulation spectroscopy, while the imino nitrogen that is directly coordinated to the Cu2+ ion is observed by pulsed electron–nuclear double resonance (ENDOR). In addition, the ENDOR spectra reveal the presence of one more nitrogen ligand that was assigned to be a deprotonated peptide nitrogen. Apart from the two nitrogen ligands, it has been established that there are two nearby hydroxyl protons, although whether these belong to a single equatorial water ligand or two equatorial hydroxide ligands is not known.
相似文献
Megan M. McEvoyEmail: Phone: +1-520-6213489Fax: +1-520-6211697 |
8.
Highly purified Na+, K+-ATPase of the dog kidney was reacted with Mg2++32Pi or Mg2++32Pi + ouabain. 32P-phosphorylation was terminated by the addition of EDTA, and the effects of various ligands on dephosphoration rate were studied. ATP reduced the dephosphorylation rates of both the native and the ouabain-complexed enzymes. K0.5 for this effect of ATP was about 0.2 mM. ADP also slowed dephosphorylation, but less effectively than ATP. The ATP effect on the native enzyme, but not that on the ouabain-complexed enzyme, was antagonized by Na+. The data establish the binding of ATP to the phosphoenzyme. Since the site that is phosphorylated by Pi is the same that is phosphorylated by ATP, coexistence of two ATP sites on the functional unit of the enzyme is suggested. 相似文献
9.
Photoluminescence and electron paramagnetic resonance properties of a potential phototherapic agent: MMgF4:Gd3+ (M = Ba,Sr) sub‐microphosphors
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Novel narrow band UVB‐emitting phosphors, BaMgF4:Gd3+ and SrMgF4:Gd3+ phosphors, were synthesized using a co‐precipitation synthesis method. X‐Ray diffraction analysis was carried out to confirm compound formation, phase purity and crystallinity of the phosphor. At 274 nm excitation, phosphors show a sharp narrow band emission at 313 nm that can be assigned to 6P7/2 → 8S7/2 transition of the Gd3+ ion. With increasing dopant concentration, intensity enhances and then decreases after a certain concentration, which is an indication of concentration quenching taking place in the phosphor. Scanning electron microscopy images of the phosphor show agglomerated particles in the sub‐micron range. Particles range in size from 600 to 800 nm. Electron paramagnetic resonance studies of the phosphors were carried out to detect radicals present in the prepared phosphor. With narrow band UVB emission, phosphor seems to be a good candidate for UV phototherapy application. Copyright © 2016 John Wiley & Sons, Ltd. 相似文献
10.
It has previously been shown that there are two sites for divalent metals at the active site of kidney (Na+ + K+)-ATPase, one bound directly to the enzyme and one coordinated to the ATP substrate [Grisham, C. (1981) J. Inorg. Biochem. 14, 45; O'Connor, S., & Grisham, C. (1980) FEBS Lett. 118, 303]. The conformation of the metal-nucleotide complex has been studied by using beta, gamma-bidentate Co-(NH3)4ATP, a substitution-inert analogue of MgATP. Kinetic studies show that Co(NH3)4ATP is a competitive inhibitor with respect to MnATP for the (Na+ + K+)-ATPase. The Ki values under both high- and low-affinity conditions (Ki = 10 microM and Ki = 1.6 mM, respectively) are similar to the Km values for MnATP under the same conditions (2.88 microM and 0.902 mM). From the paramagnetic effect of Mn2+ bound to the ATPase on the longitudinal relaxation rates of the phosphorus nuclei of Co(NH3)4ATP at the substrate site (at 40.5 and 145.75 MHz), Mn-P distances to all three phosphates are determined. The distances are consistent with the formation of a second sphere coordination complex on the enzyme between Mn2+ and the phosphates of Co(NH3)4ATP. In this respect, kidney (Na+ + K+)-ATPase appears to be similar to pyruvate kinase [Sloan, D., & Mildvan, A. (1976) J. Biol. Chem. 251, 2412] and phosphoribosylpyrophosphate synthetase [Granot, J., Gibson, K., Switzer, R., & Mildvan, A. (1980) J. Biol. Chem. 255, 10931]. Roles for both of the active site divalent cations are discussed. 相似文献
11.
On the interaction of 2,5-dibromo-3-methyl-6-isopropylbenzoquinone (DBMIB) with bound electron carriers in spinach chloroplasts 总被引:1,自引:0,他引:1
2,5-Dibromo-3-methyl-6-isopropylbenzoquinone (DBMIB), when added to chloroplasts as the sole electron donor, is an effective reducing agent. Low concentrations of 2,5-dibromo-3-methyl-6-isopropylbenzoquinone reduce cytochrome f, plastocyanin, and P700 in the dark but do not reduce the high-potential form of cytochrome b559. 2,5-Dibromo-3-methyl-6-isopropylbenzoquinone appears to interact at or near the site of function of the “Rieske” iron-sulfur center, as evidenced by a shift in the g value of the electron paramagnetic resonance signal of the reduced center. 相似文献
12.
K. Johnstone G.S.A.B. Stewart M.D. Barratt D.J. Ellar 《Biochimica et Biophysica Acta (BBA)/General Subjects》1982,714(2):379-381
The manganese electron paramagnetic spin resonance spectrum of Bacillis megaterium KM spores is virtually identical at both 77 K and 298 K which suggests that the manganese is present in a crystalline lattice in the spore core. This conclusion is supported by studies of the electron paramagnetic resonance spectra of model systems including Ca: Mn: pyridine-2,6-dicarboxylic acid crystals and manganese bound to protein and lipid standards. 相似文献
13.
Spin-labeling and multifrequency EPR spectroscopy were used to probe the dynamic local structure of skeletal myosin in the region of force generation. Subfragment 1 (S1) of rabbit skeletal myosin was labeled with an iodoacetamide spin label at C707 (SH1). X-and W-band EPR spectra were recorded for the apo state and in the presence of ADP and nucleotide analogs. EPR spectra were analyzed in terms of spin-label rotational motion within myosin by fitting them with simulated spectra. Two models were considered: rapid-limit oscillation (spectrum-dependent on the orientational distribution only) and slow restricted motion (spectrum-dependent on the rotational correlation time and the orientational distribution). The global analysis of spectra obtained at two microwave frequencies (9.4 GHz and 94 GHz) produced clear support for the second model and enabled detailed determination of rates and amplitudes of rotational motion and resolution of multiple conformational states. The apo biochemical state is well-described by a single structural state of myosin (M) with very restricted slow motion of the spin label. The ADP-bound biochemical state of myosin also reveals a single structural state (M*, shown previously to be the same as the post-powerstroke ATP-bound state), with less restricted slow motion of the spin label. In contrast, the extra resolution available at 94 GHz reveals that the EPR spectrum of the S1.ADP.Vi-bound biochemical state of myosin, which presumably mimics the S1.ADP.Pi state, is resolved clearly into three spectral components (structural states). One state is indistinguishable from that of the ADP-bound state (M*) and is characterized by moderate restriction and slow motion, with a mole fraction of 16%. The remaining 84% (M**) contains two additional components and is characterized by fast rotation about the x axis of the spin label. After analyzing EPR spectra, myosin ATPase activity, and available structural information for myosin II, we conclude that post-powerstroke and pre-powerstroke structural states (M* and M**) coexist in the S1.ADP.Vi biochemical state. We propose that the pre-powerstroke state M** is characterized by two structural states that could reflect flexibility between the converter and N-terminal domains of myosin. 相似文献
14.
The action of ATP and its analogs as well as the effects of alkali ions were studied in their action on the ouabain receptor. One single ouabain receptor with a dissociation constant (KD) of 13 nM was found in the presence of (Mg2+ + Pi) and (Na+ + Mg2+ + ATP). pH changes below pH 7.4 did not affect the ouabain receptor. Ouabain binding required Mg2+, where a curved line in the Scatchard plot appeared. The affinity of the receptor for ouabain was decreased by K+ and its congeners, by Na+ in the presence of (Mg2+ + Pi), and by ATP analogs (ADP-C-P, ATP-OCH3). Ca2+ antagonized the action of K+ on ouabain binding. It was concluded that the ouabain receptor exists in a low affinity (Rα) and a high affinity conformational state (Rβ). The equilibrium between both states is influenced by ligands of (Na+ + K+)-ATPase. With 3 mM Mg2+ a mixture between both conformational states is assumed to exist (curved line in the Scatchard plot). 相似文献
15.
Deliang Chen 《Biophysical journal》2009,96(7):2779-2788
The bacteriorhodopsin transport cycle includes protonation of the retinal Schiff base by Asp96 (M→N reaction) and reprotonation of Asp96 from the cytoplasmic surface (N→N′ reaction). We measured distance changes between pairs of spin-labeled structural elements of interest, and in general observed larger overall structural changes in the N state compared with the N′ state. The distance between the C-D loop and E-F interhelical loops in A103R1/M163R1 increased ∼6 Å in the N state and ∼3 Å in the N′ state. The opposite trend of distance changes in V101R1/A168R1 and L100R1/T170R1 supports counterclockwise rotation of helix F in the N but not the N′ state. Small distance increases were observed in S169R1/S226R1, but little change was seen in G106R1/G155R1. Taking earlier published EPR data into account, we suggest that structural changes of the E-F loop occur first, and then helices F and G begin to move together in the late M state. These motions then reach their maximum amplitude in the N state, evidently to facilitate the release of a proton from Asp96 and the formation of a proton-conduction pathway from Asp96 to the Schiff base. The structural changes reverse their directions and decay in the N′ state. 相似文献
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The in vitro reconstitution of molybdenum nitrogenase was manipulated to generate a chimeric enzyme in which the active site iron-molybdenum cofactor (FeMo-co) is replaced by NifB-co. The NifDK/NifB-co enzyme was unable to reduce N2 to NH3, while exhibiting residual C2H4 and considerable H2 production activities. Production of H2 by NifDK/NifB-co was stimulated by N2 and was dependent on NifH and ATP hydrolysis. Thus, NifDK/NifB-co is a useful tool to gain insights into the catalytic mechanism of nitrogenase. Furthermore, phylogenetic analysis of D and K homologs indicates that several early emerging lineages, which contain NifB, NifH and NifDK encoding genes but which lack other genes required for processing NifB-co into FeMo-co, might encode an enzyme with similar catalytic properties to NifDK/NifB-co. 相似文献
18.
The mechanism by which chemical energy is converted into an electrochemical gradient by P-type ATPase is not completely understood. The effects of ATP analogs on the canine kidney (Na++ K+) ATPase were compared to effects of the same analogs on the maize (Zea mays L. cv. W7551) root H+-ATPase in order to identify probes for the ATP binding site of the maize root enzyme and to determine potential similarities of ATP hydrolysis mechanisms in these two enzymes. Six compounds able to modify the ATP binding site covalently were compared. These compounds could be classed into three distinct groups based on activity. The first group had little or no effect on catalytic activity of either enzyme and included 7-chloro-4-nitrobenz-2-oxa-1.3-diazole. The second group, which included azido adenine analogs. fluorescein isothiocyanate and 5′-p-fluorosulfonylbenzoyladenine, were inhibitors of ATP hydrolysis by both enzymes. However, the sensitivity of the (Na++ K+) ATPase to inhibition was much greater than that exhibited by the maize root enzyme. The third group, which included periodate treated nucleotide derivatives and 2′,3′-o-(4-benzoylbenzoyl)adenosine triphosphate. inhibited both enzymes similarly. This initial screening of these covalent modifiers indicated that 2′,3′-o-(4-benzoylbenzoyl)adenosine triphosphate was the optimal covalent modifier of the ATP binding site of the maize root enzyme. Certain reagents were much more effective against the (Na++ K+) ATPase than the maize root enzyme, possibly indicating differences in the ATP binding and hydrolysis pathway for these two enzymes. Two ATP analogs that are not covalent modifiers were also tested: the trinitrophenyl derivatives of adenine nucleotides were better than 5′-adenylylimidodiphosphate for use as an ATP binding probe. 相似文献
19.
V D Ghanekar R J Lin R E Coffman R L Blakley 《Biochemical and biophysical research communications》1981,101(1):215-221
Exposure to nitrous oxide (N2O) is accompanied by oxidation of cob[I]alamin to the inactive cob[III]alamin [1] and to loss of methionine synthetase activity [2]. There is a steady increase in thymidylate synthetase activity in marrow collected from rats exposed to N2O and this returns to normal on restoring the animals to an air environment. 相似文献
20.
The amino acid sequence of the fluorescein isothiocyanate reactive site of lamb and rat kidney Na+- and K+-dependent ATPase 总被引:2,自引:0,他引:2
T L Kirley E T Wallick L K Lane 《Biochemical and biophysical research communications》1984,125(2):767-773
Fluorescein 5'-isothiocyanate has been used to label ouabain sensitive and insensitive (Na,K)-ATPases from lamb and rat kidney, respectively. The labeled enzymes were digested with trypsin to generate soluble peptides, which were purified by high performance liquid chromatography and sequenced on a gas phase sequenator. The sequence of the labeled peptide from both species is His-Leu-Leu-Val-Met-Lys-Gly-Ala-Pro-Glu-Arg. Thus, it appears that the primary structure of the fluorescein 5'-isothiocyanate reactive site, and therefore presumably the ATP binding site, is completely conserved in ouabain sensitive and ouabain insensitive (Na,K)-ATPases. 相似文献