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1.
Flufenamate, a non-steroidal anti-inflammatory drug, is a powerful inhibitor of anion transport in the human erythrocyte (I50 = 6·10?7M). The concentration dependence of the binding to ghosts reveals two saturable components. [14C]Flufenamate binds with high affinity (Kd1 = 1.2·10?7M) to 8.5·105 sites per cell (the same value as the number of band 3 protein per cell); it also binds, with lower affinity (Kd2 = 10?4M) to a second set of sites (4.6·107 per cell). Pretreatment of cells with 4-acetamido-4′-isothiocyanostilbene-2,2′-disulfonic acid (SITS), a specific inhibitor of anion transport, prevents [14C]flufenamate binding only to high affinity sites. These results suggest that high affinity sites are located on the band 3 protein involved in anion transport. Extracellular chymotrypsin and pronase at low concentration cleave the 95 kDa band 3 into 60 kDa and 35 kDa fragments without affecting either anion transport or [14C]flufenamate binding. Splitting by trypsin at the inner membrane surface of the 60 kDa chymotryptic fragment into 17 kDa transmembrane fragment and 40 kDa water-soluble fragment does not affect [14C]flufenamate binding. In contrast degradation at the outer membrane surface of the 35 kDa fragment by high concentration of pronase or papain decreases both anion transport capacity and number of high affinity binding sites for [14C]flufenamate. Thus it appears that 35 kDa peptide is necessary for both anion transport and binding of the inhibitors and that the binding site is located in the membrane-associated domain of the band 3 protein.  相似文献   

2.
Kinetic constants for SO42? transport by upper and lower rat ileum in vitro have been determined by computer fitting of rate vs concentration data obtained using the everted sac technique. MoO42? inhibition of this transport is competitive, and kinetic constants for the inhibition were similarly determined. Transport is also inhibited by the anions WO42?, S2O32? and SeO42?, in the order S2O32? > SeO42? ≧ MoO42? > WO42?. These anions have no effect on the transport of l-valine. Low SO42? transport rates were observed in sacs from animals fed a high-molybdenum diet. The significance of the results with respect to the problem of molybdate toxicity in animals is discussed, and related to the known protective effect of SO42?.  相似文献   

3.
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   

4.
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   

5.
With the aid of direct microfluorimetric determination of marker organic anions (fluorescein and uranin) accumulated in the proximal tubules the influence of Na+ in the bath medium on the active transport of these anions was studied. Kinetic analysis of the rate dependence of organic acid active transport into tubules on their concentration in the bath medium with a constant Na+ concentration permitted to define values of apparent Km and V for uranin and fluorescein transport in the medium with different Na+ content. It was shown that a decrease of Na+ concentration in the medium increases Km and lowers the V/Km ratio with uncharged V. By varying the Na+ concentration in the medium with a constant concentration of the marker anion the KmNa+ and VNa+ values for fluorescein and uranin transport were determined. A KmNa+ value for fluorescein in twice as much that for uranin. The 1/Km value for uranin transport is a linear function of Na+ concentration, while for fluorescein transport it is a quadratic one. Therefore it is concluded that two Na+ from the medium participate in active transfer of one fluorescein anion whereas only one Na+ from the medium is required for active transfer of one uranin anion. The run out of fluorescein from tubules preloaded with this acid is sharply reinforced by the Na+ omission from the medium. Thus, active transport of organic acids in proximal tubules of frog kidney is Na+-dependent, and Na+ from the medium is likely to participate directly in formation of a transport complex. When Na+ is absent in the medium a carrier fulfils a facilitated diffusion only.  相似文献   

6.
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   

7.
Two spectroscopic probes of free internal Ca2+ were used to determine the influence of H+ and anion permeation on the active transport of Ca2+ by skeletal sarcoplasmic reticulum. The studies were carried out on a well-characterized Ca2+-Mg2+-ATPase-rich sarcoplasmic reticulum fraction. Studies of D. McKinley and G. Meissner (1977, FEBS Lett., 82, 47–50) show that this fraction consists of two populations of vesicles: type I which has an electrically active monovalent cation (M+) permeability and type II which lacks it. The present study distinguishes between electrically active (charge-carrying) and electrically silent (e.g., countertransport) mechanisms of ion permeation in the two vesicles and shows how the active transport of Ca2+ is influenced by these permeabilities. The major results are as follows: (1) Both type I and II vesicles have an electrically active H+ permeability. (2) Type I vesicles have electrically active anion (A?) permeabilities; type II vesicles do not. (3) At low concentrations of nonpenetrating buffers, ion imbalances across the membrane can create pH imbalances. This is due to the coupling of M+ and A? movements with H+ movements. Following a jump in KCl concentration internal acidification is observed in type I vesicles while internal alkalinization is observed in type II vesicles. These pH gradients are dissipated on a time scale of seconds and tens of minutes for type I and II vesicles, respectively. (4) Tris(hydroxymethyl)aminomethane (Tris) was shown to be effective in dissipating pH gradients in type II vesicles. A model is proposed whereby HCl is equilibrated across the membrane by a Tris-catalyzed transport cycle involving transport of an ion pair between Tris-H+ and Cl? and return of the unprotonated form of the buffer. (5) The permeabilities of several physiological and nonphysiological anions were determined for type I and II vesicles. Electrically active permeability was demonstrated for Cl? and phosphate in type I vesicles. Type II vesicles lacked electrically active mechanisms for these two anions. Evidence is given for slow Cl?OH? exchange and for rapid Cl?HCO3? exchange in type II vesicles. Electrically silent phosphate influx probably occurs by H2PO4?OH? exchange. (6) Under normal conditions the Ca2+ uptake of type II vesicles is masked. It can be unmasked by addition of nigericin in the presence of Tris. The combination of ionophore and penetrating buffer render the type II vesicles KCl permeable, allowing the replenishment of internal K+ during active transport. The results are analyzed and shown to be in agreement with the Ca2+-Mg2+-ATPase pump acting as a Ca2+K+ exchanger. The results are shown to be in disagreement with electrogenic models of pump function.  相似文献   

8.
(1) H+/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 H+O, H+N2O, H+NO?2 for reduction to N2 and H+NO?2 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. H+N2O, H+NO?2 for reduction to N2 and H+NO?2 for reduction to N2O were ?0.84, ?2.33 and ?1.90, respectively. (3) The H+oxidant ratios, mentioned in item 2, were not altered in the presence of valinomycinK+ 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 H+electron acceptor ratios predicted by this scheme are in good agreement with the experimental values.  相似文献   

9.
Attention is drawn to errors common in the derivation of forms for the genotypic covariance of noninbred relatives from a Hardy-Weinberg population of diploids. A synthesis of Fisher's least-squares method of partitioning the genotypic variance and Malécot's probability method of expressing kinship, yields a general form. For one locus, the form is (Pss + Psd + Pds + Pdd) 12σa2 + (PssPdd + PsdPds) σad2, where σa2 is the additive genetic variance, αd2 is the variance of dominance deviations, pij is the probability that parental gamete i is identical by descent to parental gamete j, i = s, d indexes the parents of one relative, and j = s, d indexes those of the other. The form provides a framework for obtaining the covariance of relatives from an equilibrium population with linkage.  相似文献   

10.
11.
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   

12.
A quantitative structure-activity relationship has been formulated for 53 alkyl phosphonates [R2OPO(CH3)SR3] inhibiting chymotrypsin: log ki = 1.47MROR2 + 0.34MRSR3 + 1.25σ31 ? 1.06I ? 3.43 log (β·10MROR2 + 1) ? 5.26; log β = ?3.85. In this so-called bilinear model, ki is the bimolecular rate constant (m?1 s?1), β is a disposable parameter evaluated by a computerized iterative procedure, MR is the molar refractivity of a substituent, σ31 is Taft's polar parameter, and I is an indicator variable for substituents containing a sulfonium group. The correlation coefficient for this equation is 0.985. This quantitative structure-activity relationship is compared with those previously formulated for the action of chymotrypsin on acylamino acid ester substrates.  相似文献   

13.
The pH dependence of the reaction of tris(hydroxymethyl)aminomethane (Tris) with the activated carbonyl compound 4-trans-benzylidene-2-phenyloxazolin-5-one (I) is given by the equation k′2 = kbKa(Ka + [H+]) + ka[OH?]Ka(Ka + [H+]), where Ka is the dissociation constant of TrisH+. Spectrophotometric experiments show that the Tris ester of α-benzamido-trans-cinnamic acid is formed quantitatively over a range of pH values, regardless of the relative contribution of kb and ka terms to k2. Hence, both terms refer to alcoholysis. While the mechanism of the reaction is not determined unequivocally in the present work, the magnitude of the kb term, together with its dependence on the basic form of Tris, suggests that ester formation is occurring by nucleophilic attack of a Tris hydroxyl group on the carbonyl carbon of the oxazolinone, with intramolecular catalysis by the Tris amino group. The rate enhancement due to this group is at least 102 and possibly of the order 106. This system is compared with other model systems for the acylation step of catalysis by serine esterases and proteinases.  相似文献   

14.
The permeability of the lysosomal membrane to small anions and cations was studied at 37°C and pH 7.0 in a lysosomal-mitochondrial fraction isolated from the liver of untreated rats. The extent of osmotic lysis following ion influx was used as a measure of ion permeancy. In order to preserve electroneutrality, anion influx was coupled to an influx of K+ in the presence of valinomycin, and cation influx was coupled to an efflux of H+ using the protonophore 3-tert-butyl-5,2′-dichloro-4′-nitrosalicilylanilide. Lysosomal lysis was monitored by observing the loss of latency of two lysosomal hydrolases.The order of permeability of the lysosomal membrane to anions was found to be SCN? > I? > CH3COO? > Cl? ≈ HCO?3 ≈ Pi > SO42? and that to cations Cs+ > K+ > Na+ > H+. These orders are largely in agreement with the lyotropic series of anions and cations.The implications of these findings for the mechanism by means of which a low intralysosomal pH is produced and maintained are discussed.  相似文献   

15.
The interaction of lanthanides and other cations with phosphatidylcholine bilayers present as single bilayer vesicles in 2H2O has been investigated in terms of stoichiometry, apparent binding constants and environmental conditions.Lanthanides are shown to form 2 : 1 (molar ratio) phosphatidylcholine to metal ion complexes.The apparent binding constant Kb varies as a function of the quantity of metal ion bound and as a function of the Cl? concentration. The apparent binding constant at “zero loading” is K0 = 1.25 · 104L2 · M?at 0.15 M KCl. It decreases exponentially with increased “loading” expressed as the molar ratio of metal ion bound to effective phosphatidylcholine concentration and increases exponential with Cl? concentration.The interaction of lanthanides and divalent cations such as Ca2+ and Mg2+ is independent of pH in the pH range 3–7+ and 3–10 respectively, but is sensitive to the nature of the anion. The presence of anions enhances the interaction with polyvalent cations, the chaotropic anions showing the largest effect. The order of enhancement is Cl? < Br? < NO3? < SCN? < I? < ClO4?. The nature of the monovalent counterion (cation) has little effect on the enhanced binding of lanthanides in the presence of the above anions.The affinity of other polyvalent cations for phosphatidylcholine bilayers has been determined by competition with lanthanides. The physiologically important divalent cations Ca2+ and Mg2+ both bind less strongly (by about an order of magnitude) to the lipid surface. The order of binding of cations reflects direct binding to the phosphodiester group, with UO22+ showing the highest affinity.  相似文献   

16.
A capacitor microphone was used to measure the enthalpy and volume changes that accompany the electron transfer reactions, PQAhv P+Q?A and PQAQBhv P+QAQ?B, following flash excitation of photosynthetic reaction centers isolated from Rhodopseudomonas sphaeroides. P is a bacteriochlorophyll dimer (P-870), and QA and QB are ubiquinones. In reaction centers containing only QA, the enthalpy of P+Q?A is very close to that of the PQA ground state (ΔHr = 0.05 ± 0.03 eV). The free energy of about 0.65 eV that is captured in the photochemical reaction evidently takes the form of a substantial entropy decrease. In contrast, the formation of P+QAQ?B in reaction centers containing both quinones has a ΔHr of 0.32 ± 0.02 eV. The entropy change must be near zero in this case. In the presence of o-phenanthroline, which blocks electron transfer between Q?A and QB, ΔHr for forming P+Q?AQB is 0.13 ± 0.03 eV. The influence of flash-induced proton uptake on the results was investigated, and the ΔHr values given above were measured under conditions that minimized this influence. Although the reductions of QA and QB involve very different changes in enthalpy and entropy, both reactions are accompanied by a similar volume decrease of about 20 ml/mol. The contraction probably reflects electrostriction caused by the charges on P+ and Q?A or Q?B.  相似文献   

17.
A method for calculating the rate constant (KA1A2) 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 KA1A2 obtained by this method from that measured by the method of Parson ((1969) Biochim. Biophys. Acta 189, 384–396), namely: (4.5±1.4) · 103s?1 and (6.9±1.2) · 103 s?1, respectively.The proposed method has also been used for the estimation of the KA1A2 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 KA1A2 has been found to be (2.7±0.8) · 103 s?1.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   

18.
19.
20.
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   

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