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1.
2H and 31P spin-lattice relaxation times (T1) were studied for inverted egg phosphatidylcholine micelles in CCl4 as functions of 2H2O concentration. When the 2h2O/phosphatidylcholine mole ratio changed from 1.0 to 18.0, T1 of 31P increased by about 2.6 fold, whereas T1 of 2H increased by about 50 fold. A quantitative analysis of the deuterium T1 data showed that there is only one water molecule tightly bound to the polar head, and it is in rapid exchange with the rest of the water molecules. The activation energy for the deuterium T1 was 7.1 +/- 0.8 kcal/mol(30 +/- 3 kJ/mol), and was independent of the 2H2O concentration.  相似文献   

2.
The 14N nuclear relaxation times T1 and T2 in egg yolk phosphatidylcholine have been observed in single bilayer vesicles dispersed in the media of different viscosities, 1H2O and 2H2O. The lateral diffusion coefficient of lipid molecule D has been calculated according to the method reported earlier: D = 2.2 × 10?8cm2s?1 in 1H2O and 2.1 × 10?8cm2s?1 in 2H2O at 20°C. They are in excellent agreement. This result gives a strong basis of usefulness of 14N NMR method in the evaluation of D without introducing any system perturbation.  相似文献   

3.
Human erythrocytes were incubated in a Ringer's solution enriched with 10–18% H217O. The longitudinal relaxation time (T1) of the 17O was determined separately in samples of red cell suspesions, packed cells, and supernatant. The longitudinal relaxation of 17O in erythrocyte suspensions was non-exponential, reflecting water exchange across the cell membranes as well as relaxation processes inside and outside the cell.The T1 of intracellular 17O is 4–5 times shorter than in the supernatant, similar to the enhancement of proton relaxation by hemoglobin in erythrocytes and free solution at the frequency applied (8.13 MHz). This datum is consistent with the thesis that hemoglobin modifies the NMR relaxation behavior of water inside cells and in free solution in the same way.The rate constant
for water exchange was calculated to be 60 and 107 s−1 at 25 and at 37° C, respectively. The apparent activation energy for
over the temperature range 23–37° C was 8.7±1.0 kcal/mole.  相似文献   

4.
The steady-state rate of ATP synthesis in the isolated, Langendorff-perfused rat heart was determined using a 31P NMR saturation transfer method. At 37°C and a perfusion pressure of 70 cm H2O the value is 2.8 ± 0.3 (n=5 ± S.E.M.) μmol.s?1. (g. dry wt.)?1. The activity of creatine phosphokinase measured in the same experiments was 14.6 ± 1.0 μ mol.s?1 .(g. dry wt.)?1. From the rate of ATP synthesis and the separately measured oxygen consumption we calculated an apparent mitochondrial ADP:O ratio of 3.5 ± 0.8 in the intact tissue.  相似文献   

5.
The proton and deuterium longitudinal relaxation rates were Studied at room temperature up to the highest protein concentrations in oxyhaemoglobin solutions of different H2O/D2O composition. The deuterium relaxation rates followed the experimentally well known single linear dependence on protein concentration, the slopes being little influenced by solvent (D2O/H2O) composition. The proton ralaxation rates show two different liner dependences on haemoglobin concentration. The entire concentration range is described by two straight lines with the threshold concentration about 11 mM (in haem), The ratio of the slopes is 1.6 (high-to-low Hb-conc.). Only in the higher concentration range two T1's were observed if the solvent contained more than half of D2O. The slow relaxation phase of protons has T1's similar to those measured in solutions with less than half of D2O. The relaxation of the other phase was ten times faster. The ratio of the proton populations in these two phases was equal to 2 (slow-to-fast) and independent of protein concentration. The fast relaxing protons are attributed to water molecules encaged within two or more haemoglobin molecules which associate for times long enough on the PMR time-scale.  相似文献   

6.
We have measured the 31P n.m.r. spectra of NADP+ and NADPH in their binary complexes with Escherichia coli dihydrofolate reductase and in ternary complexes with the enzyme and folate or methotrexate. The 31P chemical shift of the 2′ phosphate group is the same in all complexes; its value indicates that it is binding in the dianionic state and its pH independence suggests that it is interacting strongly with cationic residue(s) on the enzyme. Similar behaviour has been noted previously for the complexes with the Lactobacillus casei enzyme although the 31P shift is somewhat different in this complex, possibly due to an interaction between the 2′ phosphate group and His 64 which is not conserved in the E. coli enzyme. For the coenzyme complexes with both enzymes 31POC21H2′ spin-spin interactions were detected (7.5–7.8 Hz) on the 2′ phosphate resonances, indicating a POC2H2′ dihedral angle of 30 or 330 : this is in good agreement with the value of 330° measured in crystallographic studies1 (Matthews et al., 1978) on the L. casei enzyme. NADPH-MTX complex. The pyrophosphate resonances are shifted to different extents in the various complexes and there is evidence that there is more OPO bond angle distortion in the E. coli enzyme complexes than in those with the L. casei enzyme. The effects of 31POC51H5′ spin coupling were detected on one pyrophosphate resonance and indicate that the POC5H5′ torsion angle has changed by at least ~30° on binding to the E. coli enzyme: this is considerably less than the distortion (~50°) observed previously in the L. casei enzyme complex.  相似文献   

7.
8.
Ferredoxin isolated from Halobacterium of the Dead Sea (HFd) was found to be stable and retain its conformation in 4–0.5 M salt solutions. Reconstitution of the denatured protein to the oxidized form in 2H2O indicated that the resonances shifted to the 8–10 ppm region, which include 18 protons, are nonexchangeable -NH protons. The C2H and C4H resonances of His-119 were assigned in both oxidized and reduced HFd. pH titration curves of these resonances yielded a pKa for this His of 6.57 ± 0.1 and 6.65 ± 0.1 in oxidized and reduced HFd, respectively. pH titration curves, T1 relaxation times, and the temperature dependence of the chemical shift were obtained for resonances between 6 and 10 ppm of oxidized HFd. In oxidized HFd a paramagnetically shifted resonance was observed at 15 ppm with 1 H intensity, and an anti-Curie temperature dependence. In reduced HFd eight resonances each with 1 H intensity were shifted downfield by 10–50 ppm and one resonance with 1 H intensity was shifted upfield to ?6.8 ppm. Four of these resonances exhibited an anti-Curie temperature dependence, two exhibited a moderate Curie dependence, and three were temperature independent.  相似文献   

9.
1H-, 13C-and 31P-NMR spectra of egg-yolk phosphatidylcholine (PC), phosphatidylserine (PS) and phosphatidic acid (PA) and cosonicated mixtures of these phospholipids were obtained from ultrasonicatcd dispersions containing Pr3+, Eu3+, Gd3+ and Mn2+ ions.The differences in chemical shift values. °n, between the “inner” and “outer” resonance signals for the different nuclei of the polar head group of egg-yolk phosphatidyl choline provide information about the average distances of the paramagnetic ion within the polar groups of the phospholipid molecules. In the Pr(2H2O)3+n/egg-yolk phosphatidylcholine system the ions are nearest to the phosphate and -CH2CH2 group, respectively but relatively far from the N(CH3)3 group of the polar head group of the lipid.The integral analysis of the1 H-NMR spectra obtained from dispersions containing Pr3+ and Mn2+ ions enables us to calculate the number of the polar groups in both sides of the egg-yolk phosphatidylcholine bilayer, the size of the lipid vesicle and to give some features of the arrangement of the phospholipid molecules in cosonicated egg-yolk phosphatidylcliotine/ phosphatidytserine vesicles. At p2H 8.3 in PC/PS mixtures an extreme asymmetry is observed with PS preferentially in the outer side of the membrane. This side contains approximately three times more PS than PC molecules.Some comments are made concerning the quantitative integral analysis of proton-noise decoupled 31 P-NMR spectra as obtained from similar phospholipid mixtures by Michaelson et al. and Berden et at.  相似文献   

10.
The thermotropic properties of bovine blood coagulation Factors IX and X, as well as the activation intermediates and products of these proteins, have been investigated by differential scanning microcalorimetry in the presence and absence of Ca2+. Bovine Factor IX displays a single thermal-denaturation transition characterized by a temperature midpoint (TM) of 54.5 ± 0.5 °C and a calorimetric enthalpy (ΔHc) of 105 ± 15 kcal/mol, in the absence of Ca2+. In the presence of Ca2+ concentrations sufficient to saturate its sites on Factor IX, the Tm value is increased to 57.0 ± 0.5 °C and the ΔHc is virtually unchanged. When the activation intermediate, Factor IXα, is similarly analyzed in the absence of Ca2+, a broad, diffuse thermogram was obtained which did not lend itself to calculation of thermodynamic parameters. In the presence of Ca2+, Factor IXα displayed thermograms characterized by a TM of 51.0 ± 0.5 °C and a ΔHc of 109 ± 10 kcal/mol. The activated product, Factor IXaα, in the absence of Ca2+ (the values in the presence of saturating Ca2+ are given in parentheses), undergoes thermal denaturation with a TM of 54.5 ± 0.5 °C (57.0 ± 0.5 °C) and a ΔHc of 158 ±10 kcal/mol (156 ± 10 kcal/mol). Similarly, the terminal-activation product, Factor IXaβ, displays a TM of 51.5 ± 0.5 °C (54.0 ± 0.5 °C) and a ΔHc of 85 ± 5 kcal/mol (126 ± 10 kcal/mol). Bovine blood coagulation Factor X has been analyzed in this same fashion, and shows very similar thermal properties to Factor IX. The thermal denaturation of Factor X is represented by a TM of 54.0 ± 0.5 °C (55.0 ± 0.5 °C) and a ΔHc of 102 ± 10 kcal/mol (118 ± 10 kcal/mol), whereas its activated form, Factor Xaβ, possesses a TM of 55.0 ± 0.5 °C (55.0 ± 0.5 °C) and a ΔHc of 92.0 ± 5 kcal/mol (136 ± 10 kcal/mol). These studies indicate that, for many of these proteins, Ca2+ induces a conformational alteration to a more thermally stable form, which also requires the absorption of greater amounts of heat for thermal denaturation.  相似文献   

11.
Careful experiments on the measurement of the intensity of the deuterium NMR signal for 2H2O in muscle and in its distillate were performed, and they showed that all 2H2O in muscles is “NMR visible.”The spin-lattice relaxation time (T1) of the water protons in the muscle and liver of mice and in egg white has been studied at six frequencies ranging from 4.5 to 6.0 MHz over the temperature range of +37 to −70°C. T1 values of deuterons in 2H2O of gastrocnemius muscle and liver of mice have been measured at three frequencies (4.5, 9.21 and 15.35 MHz) over the temperature range of +37 to −20°C. Calculations on T1 for both proton and deuteron have been made and compared with the experimental data. It is suggested that the reduction of the T1 values compared to pure water and the frequency dependence of T1 are due to water molecules in the hydration layer of the macromolecules, and that the bulk of water molecules in the biological tissues and egg white undergoes relaxation like ordinary liquid water.  相似文献   

12.
The anomeric composition and mutarotation rates of fructose 1,6-bisphosphate were determined in the presence of 100 mm KCl at pH 7.0 by 31P NMR. At 23 and 37 °C the solution contains (15 ± 1)% of the α anomer. The anomeric rate constants at 37 °C are (4.2 ± 0.4) s?1 for the β → α anomerization and (14.9 ± 0.5) s?1 for the reverse reaction. A D2O effect between 2.1 and 2.6 was found. From acid base titration curves it appeared that the pK values of the phosphate groups range from 5.8 to 6.0. Mg2+ and Zn2+ bind preferentially to the 1-phosphate in the α-anomeric position. Zn2+ has a higher affinity for this phosphate group than Mg2+ has. At increasing pH the fraction α anomer decreases slightly. At increasing Mg2+/fructose 1,6-bisphosphate ratios the fraction α anomer increases till 19% at a ratio of 20. Proton and probably Mg2+ binding decreases the anomerization rate. The time-averaged preferred orientation of the 1-phosphate along the C1O1 bond of the α conformer is strongly pH dependent, gauche rotamers being predominant at pH 9.4. In the presence of divalent cations the orientation is biased toward trans. A mechanistic model is proposed to explain the Zn2+, Mg2+, and pH-dependent behavior of the gluconeogenic enzyme fructose 1,6-bisphosphatase.  相似文献   

13.
A new ruthenium nitric oxide complex with the bidentate phosphine, 1,2-bis(diethylphosphino)ethane (depe), has been synthesized and characterized by UV-Vis, infrared, EPR, NMR, electrochemical techniques and X-ray structure determination. The electronic spectrum showed a typical band of dπ→pπ* charge-transfer (CT) transition, assigned to Ru(II)NO transition, and the vibrational spectrum exhibited a peak of nitrosyl ligand at (νNO=1851 cm−1). A model structure for this complex has been proposed based on 1H, 1H{31P}, 31P{1H}, 13C{1H}, COSY 1H1H{31P}, J-Resolved, HSQC, HMBC, HSQC 1H13C{31P} and 1H13C HSQC/1H1H TOCSY spectral data, and confirmed by X-ray diffraction. The nitrosonium character for the NO ligand become evident through both electron paramagnetic resonance and X-ray data (angle RuNO=177.4(3)°). The reversible monoeletronic process at E1/2=0.040 V versus SHE was assigned to the ligand NO+/NO redox couple. Under treatment with Cd(Hg) solutions containing the [Ru(NO)(depe)2Cl](PF6)2 yields a signal in the EPR spectrum (g=1.99 and g//=1.88) which fitted quite well with the simulated spectra of coordinated NO species.  相似文献   

14.
The influence of divalent cations, and pH on the behaviour of phosphatidylserine, derived from egg phosphatidylcholine, has been examined employing 31P-NMR techniques. The addition of Ca2+ results in the observation of a “rigid lattice” 31P-NMR spectra and more than an order of magnitude increase in the spin-lattice relaxation time T1. This corresponds to a strong and specific headgroup immobilization by Ca2+, similar to that observed for anhydrous phosphatidylserine. At pH 7.4 the hydrated sodium salt of (egg) phosphatidylserine adopts the bilayer phase, whereas when the pH is decreased through 3.5 a bilayer to hexagonal (HII) polymorphic phase transition is observed at 50°C, which is unaffected by equimolar cholesterol. The same transition is shown to occur at 37°C for phosphatidylserine isolated from human erythrocytes.  相似文献   

15.
NMR titration curves have been recorded for all the 13C resonances of cis and transN-acetyl-dl-proline in 2H2O. the measured pK2H values are 3.4 ± 0.8 and 4.13 ± 0.08 respectively; the free energy of ionization for the trans isomer being (3.8 kJ/mole) greater than for the cis. The ionization shifts of the two isomers differ significantly only at the acetyl carbonyl and Cγ positions. It is suggested that these are related to conformational changes which stabilize the trans form at low p2H.  相似文献   

16.
(H+ + K+)-ATPase-enriched membranes were prepared from hog gastric mucosa by sucrose gradient centrifugation. These membranes contained Mg2+-ATPase and p-nitrophenylphosphatase activities (68 ± 9 μmol Pi and 2.9 ± 0.6 μmol p-nitrophenol/mg protein per h) which were insensitive to ouabain and markedly stimulated by 20 mM KCl (respectively, 2.2- and 14.8-fold). Furthermore, the membranes autophosphorylated in the absence of K+ (up to 0.69 ± 0.09 nmol Pi incorporated/mg protein) and dephosphorylated by 85% in the presence of this ion. Membrane proteins were extracted by 1–2% (w/v) n-octylglucoside into a soluble form, i.e., which did not sediment in a 100 000 × g × 1 h centrifugation. This soluble form precipitated upon further dilution in detergent-free buffer. Extracted ATPase represented 32% (soluble form) and 68% (precipitated) of native enzyme and it displayed the same characteristic properties in terms of K+-stimulated ATPase and p-nitrophenylphosphatase activities and K+-sensitive phosphorylation: Mg2+-ATPase (μmol Pi/mg protein per h) 32 ± 9 (basal) and 86 ± 20 (K+-stimulated); Mg2+-p-nitrophenylphosphatase (μmol p-nitrophenol/mg protein per h) 2.6 ± 0.5 (basal) and 22.2 ± 3.2 (K+-stimulated); Mg2+-phosphorylation (nmol Pi/mg protein) 0.214 ± 0.041 (basal) and 0.057 ± 0.004 (in the presence of K+). In glycerol gradient centrifugation, extracted enzyme equilibrated as a single peak corresponding to an apparent 390 000 molecular weight. These findings provide the first evidence for the solubilization of (H+ + K+)-ATPase in a still active structure.  相似文献   

17.
It has been found in experiments with high resolution 31P-NMR spectroscopy (200 MHz) that the phosphocreatine peak is splitted into two different peaks in the mixtures of H2O and D2O and is single but with different chemical shifts in pure H2O and D2O. This phenomenon is explained by substitution of protons of guanidino group in phosphocreatine by deuterium. The effect of splitting disappeared at extreme pH values (>8.5 or <4.0) and at temperatures higher than 45°C due to accelerated proton-deuterium exchange. Creatine kinase added to phosphocreatine solution also lowered its temperature of peaks' collapse by 5°–10°C. A saturation (spin) transfer method was used to show that the phosphoryl group transfer to ADP in creatine kinase active center is slower with deuterium-substituted phosphocreatine than with H-phosphocreatine. The data are taken to show the importance of the proton transfer step in the creatine kinase reaction mechanism and acceleration of phosphocreatine proton-deuterium exchange by creatine kinase.  相似文献   

18.
Charcoal was found to catalyze the release of 3H2O from [1-3H]2-hydroxyestradiol-17β ([1-3H]2-OHE2) or [4-3H]2-hydroxy-estradiol-17β ([4-3H]2-OHE2) and this effect was shown to occur in the presence of glutathione or other thiols and to depend on the concentration of free steroid. The radiometric assay for measuring the formation of 3H2O was not affected significantly by subsequent treatment of the incubation mixture with charcoal if the ratio of steroid to tissue (rat brain or liver microsomes) was low and only initial rates of 3H release were measured. 2-Hydroxyestradiol did not show the charcoal effect in the presence of tyrosinase, either when it was generated from its parent estrogen or added to the enzyme. The formation of 3H2O from [4-3H]2-OHE2 in the presence of glutathione was inhibited by ascorbic acid but the addition of dextran or albumin did not protect the catechol estrogen from the charcoal-catalyzed loss of tritium. The reaction with glutathione and charcoal occurred even at 4°C but other adsorbants such as alumina, silica or hydroxylapatite were without effect.  相似文献   

19.
20.
Proton and phosphorus-31 nuclear spin–lattice relaxation times T1 and spin–spin relaxation times T2 have been measured on the single-stranded polyriboadenylic acid [poly(A)]–Mn2+ system in a neutral D2O solution in the temperature range 10°–90°C at 100 and 40.5 MHz, respectively, with the Fourier transform nmr method. Minimum values of T1 have been found for all these nuclei, which have enabled the exact estimation of apparent distances from Mn2+ to H2, H8, H1′, and the phosphorus nucleus to be 4.7, 4.1, 5.2, and 3.0 Å, respectively. The electron spin of Mn2+ penetrates into the phosphorus nucleus, giving 31P hyperfine coupling of more than 106 Hz. Evidence of penetration of the electron spin into H8 and H2 is also obtained, suggesting direct coordination of nitrogen atoms of the adenine ring to the Mn2+ Ion. Combined with the result from proton relaxation enhancement of water, it is concluded that every Mn2+ ion added is bound directly to two phosphate groups with a Mn2+–phosphorus distance of 3.3 Å, while a part of the Mn2+ ions are simultaneouly bound to the adenine ring. It is estimated that 39 ± 13% and 13 ± 5% of Mn2+ are coordinated by N7 and N3 (or N1), respectively. The motional freedom of poly(A) in the environment of the Mn2+ binding site has been found to be quenched to the extent that the rotational motion becomes several times slower than that of the corresponding Mn2+–free poly(A). The activation energies for the molecular motion are, however, practically unchanged from those for Mn2+–free poly(A), and are found to be 8.3, 8.5, 6.1, and 8.7 kcal/mol for H8, H2, H1′, and phosphorus, respectively. T2 of phosphorus is determined by the dissociation rate (k?1) of Mn2+ from the phosphate group for the whole temperature range studied with activation enthalpy of 6.5 kcal/mol. The dissociation rates of Mn2+ from the adenine ring are also estimated from proton T2 values below 50°C.  相似文献   

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