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1.
Cationic and uncharged forms of a tertiary amine local anesthetic are reported to have different properties and potencies as nerve blocking agents. However, the relative capacities of each form of the local anesthetic to perturb the properties of different model membrane systems is unknown. For this reason we have studied the effects of uncharged lidocaine (high pH) and its quaternary amine analogue (W49091) on the phase transition properties of DMPS, DPPE and DPPC liposomes using high-sensitivity differential scanning calorimetry. We report that neutral lidocaine interacts similarly with all three phospholipids. This interaction results in a decrease in the temperature of the gel å liquid crystalline phase transition (), an increase in the enthalpy of the transition (), and a slight decrease in the cooperativity of melting. Quaternary lidocaine (W49091), on the other hand, interacts significantly with only DMPS; the result being again a decrease in the temperature of DMPS melting, an increase in , and a slight decrease in the cooperativity of the phase transition. These results are interpreted to indicate that uncharged lidocaine enters the membrane during the DPPE and DPPC phase transitions. In the case of DMPS, an influx of both charged forms of lidocaine must occur at . These anesthetic fluxes at the lipid's phase transition are suggested to be responsible for the observed elevated enthalpies of the respective transitions. The observation that the cationic form of lidocaine does not significantly modify the behavior of DPPC and DPPE liposomes suggests that these lipids are not important components of the anesthetic's site in nerve membranes. However, the dramatic perturbation of the properties of DMPS by W49091 suggests that phosphatidylserine may comprise part of this inhibitory site. 相似文献
2.
A method for calculating the rate constant () for the oxidation of the primary electron acceptor (A1) by the secondary one (A2) in the photosynthetic electron transport chain of purple bacteria is proposed.The method is based on the analysis of the dark recovery kinetics of reaction centre bacteriochlorophyll (P) following its oxidation by a short single laser pulse at a high oxidation-reduction potential of the medium. It is shown that in Ectothiorhodospira shaposhnikovii there is little difference in the value of obtained by this method from that measured by the method of Parson ((1969) Biochim. Biophys. Acta 189, 384–396), namely: and , respectively.The proposed method has also been used for the estimation of the value in chromatophores of Rhodospirillum rubrum deprived of constitutive electron donors which are capable of reducing P+ at a rate exceeding this for the transfer of electron from A1 to A2. The method of Parson cannot be used in this case. The value of has been found to be .The activation energies for the A1 to A2 electron transfer have also been determined. They are 12.4 kcal/mol and 9.9 kcal/mol for E. shaposhnikovii and R. rubrum, respectively. 相似文献
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Soluble consisting predominantly of αβ-units with below 170 000 was prepared by incubating pure membrane-bound (35–48 μmol Pi/min per mg protein) from the outer renal medulla with the non-ionic detergent dodecyloctaethyleneglycol monoether (C12E8). and potassium phosphatase remained fully active in the detergent solution at C12E8/protein ratios of 2.5–3, at which 50–70% of the membrane protein was solubilized. The soluble protomeric was reconstituted to Na+, K+ pumps in phospholipid vesicles by the freeze-thaw sonication procedure. Protein solubilization was complete at C12E8/protein ratios of 5–6, at the expense of partial inactivation, but and potassium phosphatase could be reactivated after binding of C12E8 to Bio-Beads SM2. At C12E8/protein ratios higher than 6 the activities were irreversibly lost. Inactivation could be explained by delipidation. It was not due to subunit dissociation since only small changes in sedimentation velocities were seen when the C12E8/protein ratio was increased from 2.9 to 46. As determined immediately after solubilization, was 7.4 S for the fully active , 7.3 S for the partially active particle, and 6.5 S for the inactive particle at high C12E8/protein ratios. The maximum molecular masses determined by analytical ultracentrifugation were 141 000–170 000 dalton for these protein particles. Secondary aggregation occurred during column chromatography, with formation of enzymatically active or with S and apparent molecular masses in the range 273 000–386 000 daltons. This may reflect non-specific time-dependent aggregation of the detergent micelles. 相似文献
5.
The interaction of |CnH2n+1N+(CH3)3| · I? (n = 3, 6, 9, 12, 14, 16 or 18) with egg-yolk phosphatidylcholine-water dispersions has been studied by 31P-NMR spectroscopy. It is shown that the effective anisotropy of 31P chemical shift (?Δσeff) of the lamellar phospholipid liquid-crystalline phase Lα increases with increasing concentration and alkyl chain length of the drug. Addition of or |C9H19N+(CH3)3|·I? to the phospholipid-water dispersion at a molar ratio ammonium salt:phospholipid > 0.8 induces in the dispersion a structure with an effective isotropic phospholipid motion. This structure is unstable and slowly transforms into the hexagonal phase. These effects have not been observed in phospholipid-water dispersions mixed with the ammonium derivatives with the longer alkyl chains n 12, 14, 16 or 18. It is proposed that these results might explain the effects of the investigated drugs on the nerve, muscle and bacterial cells. 相似文献
6.
ADP and Pi-loaded membrane vesicles from l-malate-grown Bacillus alcalophilus synthesized ATP upon energization with . ATP synthesis occurred over a range of external pH from 6.0 to 11.0, under conditions in which the total protonmotive force was as low as ?30 mV. The phosphate potentials (ΔGp) were calculated to be 11 and 12 kcal/mol at pH 10.5 and 9.0, respectively, whereas the values in vesicles at these two pH values were quite different (?40 ± 20 mV at pH 10.5 and ?125 ± 20 mV at pH 9.0). ATP synthesis was inhibited by KCN, gramicidin, and by N,N′-dicyclohexylcarbodiimide. Inward translocation of protons, concomitant with ATP synthesis, was demonstrated using direct pH monitoring and fluorescence methods. No dependence upon the presence of Na+ or K+ was found. Thus, ATP synthesis in B. alcalophilus appears to involve a proton-translocating ATPase which functions at low . 相似文献
7.
(1) +/electron acceptor ratios have been determined with the oxidant pulse method for cells of denitrifying Paracoccus denitrificans oxidizing endogenous substrates during reduction of O2, NO?2 or N2O. Under optimal H+-translocation conditions, the ratios , , for reduction to N2 and for reduction to N2O were 6.0–6.3, 4.02, 5.79 and 3.37, respectively. (2) With ascorbate/N,N,N′,N′-tetramethyl-p-phenylenediamine as exogenous substrate, addition of NO?2 or N2O to an anaerobic cell suspension resulted in rapid alkalinization of the outer bulk medium. , for reduction to N2 and for reduction to N2O were ?0.84, ?2.33 and ?1.90, respectively. (3) The ratios, mentioned in item 2, were not altered in the presence of and the triphenylmethylphosphonium cation. (4) A simplified scheme of electron transport to O2, NO?2 and N2O is presented which shows a periplasmic orientation of the nitrite reductase as well as the nitrous oxide reductase. Electrons destined for NO?2, N2O or O2 pass two H+-translocating sites. The acceptor ratios predicted by this scheme are in good agreement with the experimental values. 相似文献
8.
The relative effectiveness of oxidizing (.OH, H2O2), ambivalent (O2?) and reducing free radicals (e? and CO2?) in causing damage to membranes and membrane-bound glyceraldehyde-3-phosphate dehydrogenase of resealed erythrocyte ghosts has been determined. The rates of damage to membranebound glyceraldehyde-3-phosphate dehydrogenase ((enz)) were measured and the rates of damage to membranes ((mb)) were assessed by measuring changes in permeability of the resealed ghosts to the relatively low molecular weight substrates of glyceraldehyde-3-phosphate dehydrogenase. Each radical was selectively isolated from the mixture produced during gamma-irradiation, using appropriate mixtures of scavengers such as catalase, superoxide dismutase and formate. .OH, O2? and H2 O2 were approximately equally effective in inactivating membrane-bound glyceraldehyde-3-phosphate dehydrogenase, while e? and CO2? were the least effective. (enz) values of O2? and H2O2 were 10-times and of .OH 15-times that of e?. (mb) values were quite similar for e? and H2O2 (about twice that of O2?), while that of .OH was 3-times that of O2?. Hence, with respect to (mb): , and with respect to (enz): . The difference between the effectiveness of the most damaging and the least damaging free radicals was more than 10-fold greater in damage to the enzyme than to the membranes. Comparison between H2O2 added as a chemical reagent and H2O2 formed by irradiation showed that membranes and membrane-bound glyceraldehyde-3-phosphate dehydrogenase were relatively inert to reagent H2O2 but markedly susceptible to the latter. 相似文献
9.
The observed equilibrium constants (Kobs) for the l-phosphoserine phosphatase reaction [EC 3.1.3.3] have been determined under physiological conditions of temperature (38 °C) and ionic strength (0.25 m) and physiological ranges of pH and free [Mg2+]. Using Σ and square brackets to indicate total concentrations . The value of Kobs has been found to be relatively sensitive to pH. At 38 °C, K+] = 0.2 m and free [Mg2+] = 0; Kobs = 80.6 m at pH 6.5, 52.7 m at pH 7.0 [ΔGobs0 = ?10.2 kJ/mol (?2.45 kcal/mol)], and 44.0 m at pH 8.0 ([H2O] = 1). The effect of the free [Mg2+] on Kobs was relatively slight; at pH 7.0 ([K+] = 0.2 m) Kobs = 52.0 m at free [Mg2+] = 10?3, m and 47.8 m at free [Mg2+] = 10?2, m. Kobs was insignificantly affected by variations in ionic strength (0.12–1.0 m) or temperature (4–43 °C) at pH 7.0. The value of K at 38 °C and I = 0.25 m has been calculated to be 34.2 ± 0.5 m [ΔGobs0 = ?9.12 kJ/mol (?2.18 kcal/ mol)]([H2O] = 1). The K for the phosphoserine phosphatase reaction has been combined with the K for the reaction of inorganic pyrophosphatase [EC 3.6.1.1] previously estimated under the same physiological conditions to calculate a value of 2.04 × 104, m [ΔGobs0 = ?28.0 kJ/mol (?6.69 kcal/mol)] for the K of the pyrophosphate:l-serine phosphotransferase [EC 2.7.1.80] reaction. . Values of Kobs for this reaction at 38 °C, pH 7.0, and I = 0.25 m are very sensitive to the free [Mg2+], being calculated to be 668 [ΔGobs0 = ?16.8 kJ/mol (?4.02 kcal/mol)] at free [Mg2+] = 0; 111 [ΔGobs0 = ?12.2 kJ/mol (?2.91 kcal/mol)] at free [Mg2+] = 10?3, m; and 9.1 [ΔGobs0 = ?5.7 kJ/mol (?1.4 kcal/mol) at free [Mg2+] = 10?2, m). Kobs for this reaction is also sensitive to pH. At pH 8.0 the corresponding values of Kobs are 4000 [ΔGobs0 = ?21.4 kJ/mol (?5.12 kcal/mol)] at free [Mg2+] = 0; and 97.4 [ΔGobs0 = ?11.8 kJ/ mol (?2.83 kcal/mol)] at free [Mg2+] = 10?3, m. Combining Kobs for the l-phosphoserine phosphatase reaction with Kobs for the reactions of d-3-phosphoglycerate dehydrogenase [EC 1.1.1.95] and l-phosphoserine aminotransferase [EC 2.6.1.52] previously determined under the same physiological conditions has allowed the calculation of Kobs for the overall biosynthesis of l-serine from d-3-phosphoglycerate. The value of Kobs for these combined reactions at 38 °C, pH 7.0, and I = 0.25 m (K+ as the monovalent cation) is 1.34 × 10?2, m at free [Mg2+] = 0 and 1.27 × 10?2, m at free [Mg2+] = 10?3, m. 相似文献
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The dielectric dispersion in the MHz range of the zwitterionic dipolar phosphocholine head groups has been measured from 0–70°C for various mixtures of (DPPC) and cholesterol. The abrupt change in the derived relaxation frequency observed for pure DPPC at the gel-to-liquid crystalline phase transition at 42°C reduces to a more gradual increase of frequency with temperature as the cholesterol content is increased. In general the presence of cholesterol increases the DPPC head group mobility due to its spacing effect. Below 42°C no sudden changes in are found at 20 or 33 mol% cholesterol, where phase boundaries have been suggested from other methods. Above 42°C, however, a decrease in at cholesterol contents up to 20–30 mol% is found. This is thought to be partly due to an additional restricting effect of the cholesterol on the number of hydrocarbon chain conformations and consequently on the area occupied by the DPPC molecules. 相似文献
12.
The changes in polymer-solvent interactions that occur when native calf thymus DNA is dialyzed against Na2SO4 solutions of a given ionic strength and buffer concentration but of varying concentrations in methylmercuric hydroxide have been investigated with the help of solution density measurements at 25 °C and pH 6.8–7.0. From measurements executed under equilibrium dialysis conditions at the three salt levels 5 mm, 0.05 m, and 0.5 m Na2SO4 (m refers to molality) and in the presence of 5 mm cacodylic acid buffer, the density increments for native calf thymus DNA were determined as a function of CH3HgOH concentration. was found not to vary with organomercurial concentration, irrespective of the concentration of supporting electrolyte, until a certain CH3HgOH concentration level has been reached, viz., , beyond which increases strongly with increasing concentration of CH3HgOH. As is shown by optical melting, becomes a function of organomercurial concentration the moment DNA undergoes denaturation brought about by the complexing of CH3HgOH with the various N-binding sites of the base residues in the DNA double helix.Polymer-solvent interactions, expressed in terms of preferential water interactions (“net hydration”) and preferential salt interactions (“salt solvation”), were derived from the data in combination with data obtained on the preferential interaction of CH3HgOH with denatured DNA and data on the partial specific volumes of all major solution components, gathered from density measurements on solutions with fixed concentrations of diffusible components. Evidence is presented which shows that denaturation in general decreases the net hydration while salt becomes preferentially associated with the polyelectrolyte. This process is further amplified by the interaction of CH3HgOH with denatured DNA: Methylmercurated DNA alters the redistribution of diffusible components at dialysis equilibrium to such an extent that in a formal sense large amounts of water are rejected from the immediate vicinity of the polymer. The molecular implications of these findings are explored. The results are further discussed in the light of previous findings where the methylmercury-induced denaturation of DNA had been studied with the help of buoyant density measurements in a Cs2SO4 density gradient and by velocity-sedimentation in a variety of sulfate media. 相似文献
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The intrinsic viscosities, weight-average molecular weights (M?w), and radii of gyration [()≈] of Streptococcus salivarius levan in various solvents were respectively obtained from viscosity and light-scattering measurements. The data showed that the levan in water is not aggregated by hydrogen bonds, and that the values of both the refractive index and () are lower in water than in aqueous solutions of urea. Urea may break intramolecular hydrogen-bonds, e.g., between branches, allowing the molecule to expand. 相似文献
15.
N-Phenylhydroxylamine is oxidized in aqueous phosphate buffer to nitrosobenzene, nitrobenzene, and azoxybenzene. Degradation is O2 dependent and shows general catalysis by (k1 = 2.3 M?2 sec?1) and (k2 = 2.3 × 105M?2 sec?1) or kinetically equivalent terms. Evidence is presented suggesting the intermediacy of a highly reactive species leading to these products. 相似文献
16.
The binding of the crustacean selective protein neurotoxin, toxin B-IV, from the nemertine Cerebratulus lacteus to lobster axonal vesicles has been studied. A highly radioactive, pharmacologically active derivative of toxin B-IV has been prepared by reaction with Bolton-Hunter reagent. Saturation binding and competition of 125I-labeled toxin B-IV by native toxin B-IV have shown specific binding of 125I-labeled toxin B-IV to a single class of binding sites with a dissociation constant of 5–20 nM and a binding site capacity, corrected for vesicle sidedness, of 6–9 pmol per mg membrane protein. This compares to a value of 3.8 pmol [3H]saxitoxin bound per mg in the same tissue. Analysis of the kinetics of toxin B-IV association () and dissociation () shows a nearly identical of about 3 nM. There is no competition of toxin B-IV binding by purified toxin from Leiurus quinquestriatus venom while Centruroides sculpturatus Ewing toxin I appears to cause a small enhancement of toxin B-IV binding. 相似文献
17.
The interactions between calmodulin, ATP and Ca2+ on the red cell Ca2+ pump have been studied in membranes stripped of native calmodulin or rebound with purified red cell calmodulin. Calmodulin stimulates the maximal rate of by 5–10-fold and the rate of Ca2+-dependent phosphorylation by at least 10-fold. In calmodulin-bound membranes ATP activates along a biphasic concentration curve (), but in stripped membranes the curve is essentially hyperbolic (). In calmodulin-bound membranes Ca2+ activates at low concentrations () in stripped membranes the apparent Ca2+ affinities are at least 10-fold lower.The results suggest that calmodulin (and perhaps ATP) affect a conformational equilibrium between E2 and E1 forms of the Ca2+ pump protein. 相似文献
18.
Chana Vinkler 《Biochemical and biophysical research communications》1981,99(4):1095-1100
The of ADP for photophosphorylation in lettuce chloroplasts was measured both at various light intensities and in the presence of various uncoupler (nigericin + K+) concentrations. Lowering the light intensity results in both, a decrease in the rate of phosphorylation and a several fold in the of ADP for the reaction. However, when increasing concentrations of the uncoupler nigericin + K+ are employed, the rate of photophosphorylation is decreased but a several-fold in the of ADP for the reaction is observed. The results are discussed in terms of the chemiosmotic hypothesis. It is suggested that these effects might indicate the existence of a mechanism controlling the rate of ATP formation which is different than the formation of the electrochemical gradient. 相似文献
19.
Nuclear magnetic resonance study of the rate of electron transfer between cytochrome c and iron hexacyanides 总被引:5,自引:0,他引:5
The rates of electron exchange between ferricytochrome c (CIII)3 and ferrocytochrome c (CII) were observed as a function of the concentrations of ferrihexacyanide (FeIII) and ferrohexacyanide (FeII) by monitoring the line widths of several proton resonances of the protein. Addition of FeII to CIII homogeneously increased the line widths of the two downfield paramagnetically shifted heme methyl proton resonances to a maximal value. This was interpreted as indicating the formation of a stoichiometric complex, CIII·FeII, in the over-all reaction: Values for , respectively, were calculated from the maximal change in line width observed at pH 7.0 and 25 °C. Changes in the line width of CIII in the presence of FeII and either KCl or FeIII suggest that complexation is principally ionic, that FeIII and FeII compete for a common site. Addition of saturating concentrations of FeIII to CIII produced only minor changes in the nuclear magnetic resonance spectrum of CIII suggesting that complexation occurs on the protein surface.Addition of FeIII to CII in the presence of excess FeII (to retain most of the protein as CII) increased the line width of the methyl protons of ligated methionine 80. A value for k?2 ≈ 2.08 × 104 s?1 was calculated from the dependence of linewidth on the concentration of FeII at 24 °C. These rates are shown to be consistent with the over-all rates of reduction and oxidation previously determined by stopped flow measurements, indicating that k2 and k?2 were rate limiting. From the temperature dependence the enthalpies of activation are 7.9 and 15.2 kcal/mol for k2 and k?2, respectively. 相似文献
20.
J K Ladha P Rowell W D Stewart 《Biochemical and biophysical research communications》1978,83(2):688-696
5-hydroxylysine, an analogue of glutamate and lysine, causes production by N2-fixing A. cylindrica; it also reversibly inhibits GS activity in vitro but has no effect on alanine dehydrogenase or GOGAT. On adding 5-hydroxylysine intracellular pools of glutamine, glutamate and aspartate decrease; those of alanine and serine increase. 5-hydroxylysine alleviates the inhibitory effect of on heterocyst production and C2H2 reduction and in cultures results in heterocyst synthesis and in C2H2 reduction. The data suggest that the GS-GOGAT pathway is the sole route of importance in primary assimilation in A. cylindrica, that alone does not inhibit nitrogenase and heterocyst production, and that GS and/or a product is involved in regulating the production of both. 相似文献