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1.
Using guanidinium and n-butylammonium cations (C+) as models for the positively charged side chains in arginine and lysine, we have determined the association constants with various oxyanions by potentiometric titration. For a dibasic acid, H2A, three association complexes may exist: K1M = [CHA][C+] [HA?]; K1D = [CA?][C+] [A2?]; K2D = [C2A][C+] [CA?]. For guanidinium ion and phosphate, K1M = 1.4, K1D = 2.6, and K2D = 5.1. The data for carboxylates indicate that the basicity of the oxyanion does not affect the association constant: acetate, pKa = 4.8, K1M = 0.37; formate, pKa = 3.8, K1M = 0.32; and chloroacetate, pKa = 2.9, K1M = 0.43, all with guanidinium ion. Association constants are also reported for carbonate, dimethylphosphinate, benzylphosphonate, and adenylate anions.  相似文献   

2.
The rate of reaction of [Cr(III)Y]aq (Y is EDTA anion) with hydrogen peroxide was studied in aqueous nitrate media [μ = 0.10 M (KNO3)] at various temperatures. The general rate equation, Rate = k1 + k2K1[H+]?11 + K1[H+]?1 [Cr(III)Y]aq[H2O2] holds over the pH range 5–9. The decomposition reaction of H2O2 is believed to proceed via two pathways where both the aquo and hydroxo-quinquedentate EDTA complexes are acting as the catalyst centres. Substitution-controlled mechanisms are suggested and the values of the second-order rate constants k1 and k2 were found to be 1.75 × 10?2 M?1 s?1 and 0.174 M?1 s?1 at 303 K respectively, where k2 is the rate constant for the aquo species and k2 is that for the hydroxo complex. The respective activation enthalpies (ΔH*1 = 58.9 and ΔH*2 = 66.5 KJ mol?1) and activation entropies (ΔS*1 = ?85 and ΔS*2 = ?40 J mol?1 deg?1) were calculated from a least-squares fit to the Eyring plot. The ionisation constant pK1, was inferred from the kinetic data at 303 K to be 7.22. Beyond pH 9, the reaction is markedly retarded and ceases completely at pH ? 11. This inhibition was attributed in part to the continuous loss of the catalyst as a result of the simultaneous oxidation of Cr(III) to Cr(VI).  相似文献   

3.
Na+, K+ and Cl? concentrations (cji) and activities (aji), and mucosal membrane potentials (Em) were measured in epithelial cells of isolated bullfrog (Rana catesbeiana) small intestine. Segments of intestine were stripped of their external muscle layers, and bathed (at 25°C and pH 7.2) in oxygenated Ringer solutions containing 105 mM Na+ and Cl? and 5.4 mM K+. Na+ and K+ concentrations were determined by atomic absorption spectrometry and Cl? concentrations by conductometric titration following extraction of the dried tissue with 0.1 M HNO3. 14C-labelled inulin was used to determine extracellular volume. Em was measured with conventional open tip microelectrodes, aCli with solid-state Cl?-selective silver microelectrodes and aNai and aKi with Na+- and K+-selective liquid ion-exchanger microelectrodes. The average Em recorded was ?34 mV. cNai, cKi and cCli were 51, 105 and 52 mM. The corresponding values for aNai, aKi and aCli were 18, 80 and 33 mM. These results suggest that a large fraction of the cytoplasmic Na+ is ‘bound’ or sequestered in an osmotically inactive form, that all, or virtually all the cytoplasmic K+ behaves as if in free solution, and that there is probably some binding of cytoplasmic Cl?. aCli significantly exceeds the level corresponding to electrochemical equilibrium across the mucosal and baso-lateral cell membranes. Earlier studies showed that coupled mucosal entry of Na+ and Cl? is implicated in intracellular Cl? accumulation in this tissue. This study permitted estimation of the steady-state transapical Na+ and Cl? electrochemical potential differences (Δμ̄Na and Δμ̄Cl). Δμ̄Na (?7000 J · mol?1; cell minus mucosal medium) was energetically more than sufficient to account for Δμ̄Cl (1000–2000 J · mol?1).  相似文献   

4.
5.
N-Phenylhydroxylamine is oxidized in aqueous phosphate buffer to nitrosobenzene, nitrobenzene, and azoxybenzene. Degradation is O2 dependent and shows general catalysis by H2PO4? (k1 = 2.3 M?2 sec?1) and PO4?3 (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   

6.
Analysis of the cation composition of growing Mycoplasma mycoides var. Capri indicates that these organisms have a high intracellular K+ concentration (Ki: 200–300 mM) which greatly exceeds that of the growth medium, and a low Na+ concentration (Nai+: 20 mM). Unlike Nai+, Ki+ varies with cell aging.The K+ transport properties studied in washed organisms resuspended in buffered saline solution show that cells maintain a steady and large K+ concentration gradient across their membrane at the expense of metabolic energy mainly derived from glycolysis. In starved cells, Ki+ decreases and is partially compensated by a gain in Na+. This substitution completely reverses when metabolic substrate is added (K+ reaccumulation process). Kinetic analysis of K+ movement in cells with steady K+ level shows that most of K+ influx is mediated by an autologous K+-K+ exchange mechanism. On the other hand, during K+ reaccumulation by K+-depleted cells, a different mechanism (a K+ uptake mechanism) with higher transport capacity and affinity drives the net K+ influx. Both mechanisms are energy-dependent.Ouabain and anoxia have no effect on K+ transport mechanisms; in contrast, both processes are completely blocked by dicyclohexylcarbodiimide, an inhibitor of the Mg2+-dependent ATPase activity.  相似文献   

7.
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:
CIII+FeII?k?1k1CIII·FeII?k?2k2CII·FeIII?k?3k3CIII+FeII
Values for k1k?1 = 0.4 × 103m?1and k2 = 208 s?1, 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   

8.
(1) A quantitative study has been made of the binding of ouabain to the (Na+ + K+)-ATPase in homogenates prepared from brain tissue of the hawk moth, Manduca sexta. The results have been compared to those obtained in bovine brain microsomes. (2) The insect brain (Na+ + K+)-ATPase will bind ouabain either in the presence of Mg2+ and Pi, (‘Mg2+, Pi’ conditions) or in the presence of Na+, Mg2+, and an adenine nucleotide (‘nucleotide’ conditions) as is the case for the bovine brain (Na+ + K+)-ATPase. The binding conditions did not alter the total number of receptor sites measured at high ouabain concentrations in either tissue. (3) Potassium ion decreases the affinity (increases the KD) of ouabain to the M. sexta brain (Na+ + K+)-ATPase under both binding conditions. However, ouabain binding is more sensitive to K+ inhibition under the nucleotide conditions. In bovine brain ouabain binding is equally sensitive to K+ inhibition under the both conditions. (4) The enzyme-ouabain complex has a rate of dissociation that is 10-fold faster in the M. sexta preparation than in the bovine brain preparation. Because of this, the M. sexta (Na+ + K+)-ATPase has a higher KD for ouabain binding and is less sensitive to inhibition by ouabain than the bovine brain enzyme. (5) This data supports the hypothesis that two different conformational states of the M. sexta (Na+ + K+)-ATPase can bind ouabain.  相似文献   

9.
Presteady-state kinetic studies of α-chymotrypsin-catalyzed hydrolysis of a specific chromophoric substrate, N-(2-furyl)acryloyl-l-tryptophan methyl ester, were performed by using a stopped-flow apparatus both under [E]0 ? [S]0 and [S]0 ? [E]0 conditions in the pH range of 5–9, at 25 °C. The results were accounted for in terms of the three-step mechanism involving enzyme-substrate complex (E · S) and acylated enzyme (ES′); no other intermediate was observed. This substrate was shown to react very efficiently, i.e., the maximum of the second-order acylation rate constant (k2Ks)max = 4.2 × 107 M?1 s?1. The limiting values of Ks′ (dissociation constant of E · S), K2 (acylation rate) and k3 (deacylation rate) were obtained from the pH profiles of these parameters to be 0.6 ± 0.2 × 10?5 m, 360 ± 15 s?1 and 29.3 ± 0.8 s?1, respectively. Likewise small values were observed for Ki of N-(2-furyl)-acryloyl-l-tryptophan and N-(2-furyl)acryloyl-d-tryptophan methyl ester and Km of N-(2-furyl)acryloyl-l-tryptophan amide. The strong affinities observed may be due to intense interaction of β-(2-furyl)acryloyl group with a secondary binding site of the enzyme. This interaction led to a k?1k2 value lower than unity, i.e., the rate-limiting process of the acylation was the association, even with the relatively low k2 value of this methyl ester substrate, compared to those proposed for labile p-nitrophenyl esters.  相似文献   

10.
Quercetin inhibited a dog kidney (Na+ + K+)-ATPase preparation without affecting Km for ATP or K0.5 for cation activators, attributable to the slowly-reversible nature of its inhibition. Dimethyl sulfoxide, a selector of E2 enzyme conformations, blocked this inhibition, while the K+-phosphatase activity was at least as sensitive to quercetin as the (Na+ + K+)-ATPase activity, all consistent with quercetin favoring E1 conformations of the enzyme. Oligomycin, a rapidly-reversible inhibitor, decreased the Km for ATP and the K0.5 for cation activators, and its inhibition was also diminished by dimethyl sulfoxide. Although oligomycin did not inhibit the K+-phosphatase activity under standard assay conditions, a reaction presumably catalyzed by E2 conformations, its effects are nevertheless accommodated by a quantitative model for that reaction depicting oligomycin as favoring E1 conformations. The model also accounts quantitatively for effects of both dimethyl sulfoxide and oligomycin on Vmax, Km for substrate, and K0.5 for K+, as well as for stimulation of phosphatase activity by both these reagents at low K+ but high Na+ concentrations.  相似文献   

11.
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 Kobs = Σ L-serine][Σ Pi]Σ L-phosphoserine]H2O], K = L-H · serine±]HPO42?][L-H · phosphoserine2?]H2O]. 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. Kobs = [Σ L-serine][Σ Pi][Σ L-phosphoserine][H2O], K = [L-H · serine±]HPO42?][L-H · phosphoserine2?]H2O. 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. Kobs = [Σ L-serine][Σ NADH][Σ Pi][Σ α-ketoglutarate][Σ d-3-phosphoglycerate][Σ NAD+][Σ L-glutamat0] 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   

12.
Proton inventory investigations of the hydrolysis N-acetylbenzotriazole at pH 3.0 (or the equivalent point on the pD rate profile) have been conducted at two different temperatures and at ionic strengths ranging from 0 to 3.0 M. The solvent deuterium isotope effects and proton inventories are remarkably similar over this wide range of conditions. The proton inventories suggest a cyclic transition state involving four protons contributing to the solvent deuterium isotope effect for the water-catalyzed hydrolysis. The hydrolysis data are described by the equation kn = ko (1 ? n + nπa1)4 with πa1 ~ 0.74, where ko is the observed first-order rate constant in protium oxide, n is the atom fraction of deuterium in the solvent, kn is the rate constant in a protium oxide-deuterium oxide mixture, and πa1 is the isotopic fractionation factor.  相似文献   

13.
The technique of laser Doppler electrophoresis was applied for the study of the surface charge properties of (Na+,+)-ATPase containing microsomal vesicles derived from guinea-pig kidney. The influence of pH, the screening and binding of uni- and divalent cations and the binding of ATP show: (1) one net negative charge per protein unit with a pK = 3.9; (2) deviation from the Debye relation between surface potential and ionic strength for univalent cations, with no difference in the effect of Na+ and K+; (3) Mg2+ binds with an association constant of Ka = 1.1 · 102M?1 while ATP binds with an apparent Ka = 1.1 · 104M?2 for 1 mM Nacl, 0.2 mM KCI, 0.1 mM MgCl2, 0.1 mM Tris-HCI (pH 7.3). The binding is weaker at higher Mg2+ concentrations. There is no ATP binding in the absence of Mg2+. In addition, the average vesicle size derived from the linewidth of the quasi-elastic light scattering spectrum is 203.7 ± 15.2 nm. In the presence of ATP a reduction in size is observed.  相似文献   

14.
(1) A membrane fraction enriched in (Na+ + K+)-ATPase (EC 3.6.1.3) was obtained from optic ganglia of the squid (Loligo pealei) by density gradient fractionation of membranes followed by treatment with either SDS or Brij-58. The resulting membrane had an (Na+ + K+)-ATPase specific activity of approx. 2 units/mg and was >95% ouabain-sensitive. (2) The (Na+ + K+)-ATPase had a Km for ATP of 0.42 ± 0.04 mM and a pH optimum of 7.0. It was inhibited by ouabain with a Ki of 0.32 ± 0.04 μM. (3) Optimum monovalent cation concentrations were: 240 mM NaCl, 60 mM KCl, tested with NaCl + KCl = 300 mM. (4) The Mg2+ dependence of hydrolysis varied with the absolute ATP concentration. At 3 mM ATP, theKm for Mg2+ was 0.86 ± 0.10 mM, and at 6 mM ATP, the Km was 1.86 ± 0.44 mM. High levels of Mg2+ caused inhibition of hydrolysis. (5) The interactions of Na+ and K+ were examined over a range of conditions. K+ levels caused modulations in the Na+ dependence in the range of 1–150 mM. (6) The (Na+ + K+)-ATPase prepared from squid optic ganglion displays properties similar to those of the sodium pump in injected nerves.  相似文献   

15.
The kinetics of isotopic Na+ flows was studied in urinary bladders of toads from the Dominican Republic. Initial studies of the potential dependence of passive serosal to mucosal 22Na+ efflux demonstrated the absence of isotope interaction and/or other coupling with passive Na+ flow. The electrical current I and mucosal to serosal 22Na+ influx were then measured with transmembrane potential clamped at Δψ = 0, 25, 50, 75 or 100 mV. Subsequent elimination of active Na+ transport mucosal amiloride permitted calculation of the rates of active Na+ transport JNaa and active and passive influx JNaNa and JNaa and JNap. The results indicate that for Dominican toad bladders mounted in chambers only Na+ contributes significantly to transepithelial active ion transport; hence JNaa = Ja. Ja was abolished at Δψ = E = 96.3 ± 1.9 (S.E.) mV. As Δψ approached E, active efflux Ja became demonstrable. At Δ = 100 mV, Ja exceeded Ja, so that Ja was negative. Experimental values of Ja agreed well with theoretical values predicted by a thermodynamic formulation: Jexpa = 0.985 Jtheora (r = 0.993). The dependence of Ja on Δψ is curvilinear.  相似文献   

16.
17.
A maximal rate of the ouabain-sensitive 204Tl influx in human erythrocytes can be attained at trace concentrations of Tl+ in Mg2+ isotonic media free of K+ and Na+. The maximal influx of Tl+ from isotonic Mg(NO3)2 at 20°C and pH 7.4 was 0.45 mM · 1?1 · h?1 with a Km of 0.025 mM. In contrast to the active influx of Tl+, the passive Tl+ fluxes were neither saturated nor influenced by external cations in the range of concentrations of Tl+ and K+ studied. The rate constants of Tl+ passive fluxes in human and cat erythrocytes can be related to pH by the equation log kin(out) = –A + B · pH, where A and B are empirical constants for particular conditions. The apparent activation energy was 16 and 11 kcal/mol in sulphate and nitrate media, respectively. Tl+ and the alkali metal cations seem to overcome a common barrier in the erythrocyte membrane. Nevertheless, the rate of the passive penetration of Tl+ is about two orders of magnitude faster than those of K+ or Rb+. An extra non-Coulombic interaction between Tl+ and membrane ligands appears to be involved providing an accumulation of Tl+ somewhere in the vicinity of the membrane barrier and increasing the diffusion fluxes of Tl+ in both directions.  相似文献   

18.
The active transport of neutral amino acids into Streptomyces hydrogenans is inhibited by external Na+. There is no indication that in these cells amino acid accumulation is driven by an inward gradient of Na+. The extent of transport inhibition by Na+ depends on the nature of the amino acid. It decreases with increasing chain length of the amino acid molecules i.e. with increasing non-polar properties of the side chain. Kinetic studies show that Na+ competes with the amino acid for a binding site at the amino acid carrier. There is a close relation between the Ki values for Na+ and the number of C atoms of the amino acids. Other cations also inhibit neutral amino acid uptake competitively; the effectiveness decreases in the order Li+ > Na+ > K+ > Rb+ > Cs+. Anions do not have a significant effect on the uptake of neutral amino acids. After prolonged incubation of the cells with 150 mM Na+, in addition to the competitive inhibition of transport Na+ induces an increase in membrane permeability for amino acids.  相似文献   

19.
The kinetics of bisulfite addition to 5-fluorouracil were studied as a function of increasing concentrations of potential general acids. Values of kobsd[SO3=] measured at 25°C and ionic strength 1.0 M increased linearly and then became invariant with increasing concentrations of either HSO3? or (OHCH2CH2)2N+C(CH2OH)3 HCl (BisTris+HCl). A small kinetic hydrogen-deuterium isotope effect (kHSkDS = 1.10) was observed for the general acid catalysed portion of the addition reaction. The kinetics of bisulfite elimination from 5-fluoro-5,6-dihydrouracil-6-sulfonate were studied in ethanolamine buffers. As previously observed with 1,3-dimethyl-5,6-dihydrouracil-6-sulfonate, this reaction is subject to general base catalysis and exhibits a large kinetic hydrogen-deuterium isotope effect (k2H2Ok2D2O = 3.8). The kinetic results for the addition reaction are consistent with a multistep reaction pathway involving the initial formation of an oxyanion sulfite addition intermediate (II) which subsequently adds a proton and undergoes tautomerization to yield the final 5-fluoro-5,6-dihydrouracil-6-sulfonate product. Thus the elimination of bisulfite from 5-fluoro-5,6-dihydrouracil-6-sulfonate probably proceeds by an ElcB mechanism which involves, at relatively low concentrations of general base, rate determining general base catalyzed proton abstraction from carbon 5 to yield intermediate II followed by the rapid elimination of sulfite to yield 5-fluorouracil. These results may be related to both the enzymatically catalyzed dehalogenation of bromoand iodouracil and the methylation of deoxyuridylate by thymidylate synthetase.  相似文献   

20.
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