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1.
The nuclear magnetic quadrupole relaxation enhancement of 35Cl-, 81Br-, and 12I- anions on binding to human serum albumin has been studied under conditions of variable protein and anion concentration and also in the presence of simple inorganic, amphiphilic, and complex anions which compete with the halide ions for the protein anion binding sites. Two classes of anion binding sites with greatly different binding constans were identified. Experiments at variable halide ion concentration were employed to determin the Cl- and I- binding constants. By means of 35 Cl nuclear magnetic resonance (NMR) the relative affinity for different anions was determined by competition experiments for both the strong and the weak anion binding sites. Anion binding follows the sequence SO42- smaller than F- smaller than CH3COO- smaller than Ci- smaller Br- smaller than NO3- smaller than I- smaller than ClO4- smaller than SCN- smaller than Pt(CN)42- smaller than Au(CN)2- smaller than CH3(CH2)11OSO3- for the high affinity sites, and the sequence SO42- congruent to F- congruent to Cl- smaller CH3COO- smaller than NO3- smaller than Br- smaller than I- smaller than ClO4- smaller than SCN- for the low affinity sites. These series are nearly identical with the well-known lyotropic series. Consequently, those effects of anions on proteins described by the lyotropic series can be correlated with the affinities of the anions for binding to the protein. The data suggest that the physical nature of the interaction is the same for both types of biding sites, and that the differences in affinity between different binding sites must be explained in terms of tertiary structure. Analogous experiments performed using 127I- quadrupole relaxation gave results very similar to those obtained with 35Cl-. A comparison between the Cl-, Br- and I- ions revealed that, as a result of the increasing affinity for the weak anion binding sites in the series Cl- smaller than Br- smaller than I-, Cl- is much more useful as a probe for the specific anion binding sites than the other two halide ions. The findings with human serum albumin in this and other respects are probably of general relevance in studies of protein-anion interactions. In addition to competition experiments, the magnitude of the relaxation rate is also discussed. Line broadening not related to anion binding to the protein is found to be small. A comparison of transverse and longitudinal 35Cl relaxation rates gives a value for the quadrupole coupling constant of the high affinity sites in good agreement with a calculated coupling constant assuming anion binding to arginine. 相似文献
2.
The interaction of proteins with hydroxyapatite. I. Role of protein charge and structure 总被引:5,自引:0,他引:5
M J Gorbunoff 《Analytical biochemistry》1984,136(2):425-432
The criteria for elution of proteins from hydroxyapatite columns were examined as a function of (1) protein isoelectric point (22 proteins with isoelectric points between 3.5 and 11.0); (2) ionic nature of eluant (Na salts of PO4, F-, Cl-, SCN-, ClO-4, and CaCl2); and (3) structural differences between related proteins. It was found that proteins can be classified into three groups: (1) basic proteins, which elute at similar, moderate molarities of PO4, F-, Cl-, SCN-, and ClO-4, and low (less than 0.003 M) Ca2+; (2) acidic proteins which elute at about equal moderate molarities of PO4 and F-, but do not elute with Ca2+ and usually not with Cl-; (3) neutral proteins, which elute with PO4, F-, and Cl-, but show a strong anion specificity, and do not elute with Ca2+ or SCN-. Furthermore, individual specific polar groups are not in general crucial to binding or desorption, and variations in structure, other than major loosening, do not influence strongly the pattern of protein-hydroxyapatite interaction. 相似文献
3.
Lysis of Streptococcus mutans by hen egg white lysozyme and inorganic sodium salts. 总被引:8,自引:2,他引:6
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H Goodman J J Pollock L I Katona V J Iacono M I Cho E Thomas 《Journal of bacteriology》1981,146(2):764-774
Streptococcus mutans BHT was grown in a synthetic medium containing radioactive thymidine to monitor deoxyribonucleic acid release. Kinetic experiments demonstrated that although lysozyme alone could not liberate deoxyribonucleic acid, cellular deoxyribonucleic acid was liberated from lysozyme-treated cells by addition of low concentrations of inorganic sodium salts. When the salts were tested for their ability to dislodge cell-bound tritiated lysozyme, the extent of the initial release of enzyme by individual anions correlated with the anion potency for deoxyribonucleic acid liberation (SCN- greater than ClO4- greater than I- greater than Br- greater than NO3- greater than Cl- greater than F-), although the total amount of lysozyme dislodged did not correspond directly with cell lysis. Differences in the effectiveness of anions (SCN-, HCO3-, Cl- and F-) in potentiating cell lysis could be enhanced or minimized by varying the lysozyme, anion, and bacterial cell concentrations. As the anion concentration was increased for each enzyme concentration and cell concentration, the lysis increased, in some cases markedly, until maximum levels of released deoxyribonucleic acid were attained. The maximum levels of lysis of SCN- and HCO3- were similar and were greater than those for Cl- and F-. In addition, the maximum levels were observed to increase for each of the anions as the concentration of lysozyme increased. 相似文献
4.
The effectiveness of various salts of the Hofmeister series as dissociating agents for human hemoglobin A tetramers has been investigated by light-scattering molecular-weight measurements. Dissociation of hemoglobin to half-molecules of alpha beta dimers follows the order of the series dictated predominantly by the sequence of the anions F- less than Cl- less than Br- less than ClO4- less than SCN-, I-, with the cations Na+ and K+ having relatively little effect on the observed dissociation. The use of equations derived for predicting the effects of dissociating reagents on the structure of subunit proteins [Herskovits, T. T., and Ibanez, V. S. (1976), Biochemistry 15, 5715] together with Setschenow constants based on the model amino acid data of Nandi and Robinson were found to give a satisfactory account of the dissociation behavior observed with many of the salts, giving reasonable estimates of the number of amino acids that form the smaller contact area of the alpha beta subunits of hemoglobin shown by the Perutz crystallographic model. The analysis of the dissociation data also extends the utility of the Setschenow constants tested for the characterization of the dissociation behavior of other subunit proteins. 相似文献
5.
The accessibility of protein-bound dinitrophenyl groups to univalent fragments of anti-dinitrophenyl antibody. 总被引:1,自引:1,他引:0
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A series of N-(N-dinitrophenylaminoalkyl)maleimides were sythesized with alkyl-chain lengths of two, four and six carbon atoms. When these compounds reacted with the thiol group of mercaptalbumin, the tryptophan fluorescence of the protein was quenched. This change in fluorescence was used to determine the rate of reaction of the Dnp (dinitrophenyl)-maleimides with mercaptalbumin. The second-order rate constants were similar to those observed in reactions between low-molecular-weight thiol compounds and maleimides. When N-(N-Dnp-aminoalkyl)succinimidomercaptalbumins were added to univalent fragments of anti-Dnp antibody the antibody fluorescence was quenched. Florescence-quenching titrations showed that the protein-bound Dnp groups were fully available to the antibody even when the alkyl chain was short. The apparent dissociation constants were significantly greater than that of the interaction between anti-Dnp antibody and the free hapten, 6-(N-Dnp)-aminohexanoate. The antibody fluorescence was quenched efficienty by [dnp-Lys41]ribonuclease A, also with an increased dissociation constant. It could be concluded from the increase in dissociation constant that the Dnp group spent no more than 0.1% of its time in the dissociated state, available to antibody. The second-order rate constants for the association between the Dnp-mercaptablumins and the antibody were determined and were similar in magnitude to those observed in other interactions between protein and anti-protein antibody. 相似文献
6.
Recently we have demonstrated that the exposure of monomeric Cd7-metallothionein (MT) to Cd(II) ions in potassium phosphate buffer results in the nonoxidative formation of MT dimers containing approximately two additional Cd(II) ions/monomer subunit [Palumaa, P., Mackey, E. and Vasák, M. (1992) Biochemistry 31, 2181-2186]. In this study, we demonstrate that inorganic phosphate participates in the Cd-induced dimerization of MT. In the absence of phosphate, Cd-induced oligomerization of MT still takes place, but a substantially lower apparent yield of the dimeric form and an additional peak of MT tetramers were detected in gel-filtration experiments. Arsenate exhibits a similar effect to that of phosphate, whereas a number of other anions, i.e. F-, NO3-, SO4(2-), ClO4-, BO3-, SCN-, HCOO- and CH3COO- had no effect on Cd-induced oligomerization of MT. Studies on the pH dependence of MT dimerization indicate that the dianionic form of phosphate is involved in this process. Equilibrium-dialysis experiments using potassium [32P]phosphate established binding of two molecules of phosphate to the dimeric MT form with a dissociation constant, Kd, of 23 +/- 3 microM (20 mM Tris/HCl and 0.1 M KCl, pH 8.0 at 25 degrees C), whereas binding of phosphate was not observed with the monomeric Cd7-MT. The noncovalent nature of phosphate binding to the Cd-induced MT dimers has been demonstrated. The presented data provide the first evidence for the binding of a nonmetallic cellular component to MT. 相似文献
7.
Myeloperoxidase (MPO) structural analysis has suggested that halides and pseudohalides bind to the distal binding site and serve as substrates or inhibitors, while others have concluded that there are two separate sites. Here, evidence for two distinct binding sites for halides comes from the bell-shaped effects observed when the second-order rate constant of nitric oxide (NO) binding to MPO was plotted versus Cl- concentration. The chloride level used in the X-ray structure that produced Cl- binding to the amino terminus of the helix halide binding site was insufficient to populate either of the two sites that appear to be responsible for the two phases. Biphasic effects were also observed when the I-, Br-, and SCN- concentrations were plotted against the NO combination rate constants. Interestingly, the trough concentrations obtained from the bell-shaped curves are comparable to normal plasma levels of halides and pseudohalides, suggesting the potential relevance of these molecules in modulating MPO function. The second-order rate constant of NO binding in the presence of plasma levels of I-, Br-, and SCN- is 1-2-fold lower compared to that obtained in the absence of these molecules and remains unaltered through the Cl- plasma level. When Cl- exceeded the plasma level, the NO combination rate becomes indistinguishable from the second phase of the bell-shaped curve that was obtained in the absence of halides. Our results are consistent with two halide binding sites that could be populated by two halides in which both display distinct effects on the MPO heme iron microenvironment. 相似文献
8.
Monoclonal antibody to rat apoC: multiple binding to apoC-I on lipoproteins increases its affinity constant 总被引:1,自引:0,他引:1
L Wong 《Journal of lipid research》1985,26(7):790-796
Using solid phase systems, the kinetics of binding of monoclonal antibody (LRB 45, IgG2b,kappa) to apoC-I and apoC-I on lipoproteins were investigated. At 25 degrees C, the association constant of LRB 45 antibody to apoC-I (3.56 X 10(6) M-1 X sec-1) was almost three times slower than the association constant LRB 45 antibody to lipoproteins (10.4 X 10(6) M-1 X sec-1). However, the dissociation constant of apoC-I from LRB 45 antibody (0.865 X 10(-4) sec-1) was also slower than the dissociation constant of lipoprotein from antibody (1.5 X 10(-4) sec-1). Thus, the calculated affinity constant (association constant/dissociation constant) of LRB 45 antibody for apoC-I was approximately half of that for lipoproteins (4.12 X 10(10) M-1 vs. 6.92 X 10(10) M-1). The intrinsic affinity constants for antibody binding to apoC-I and apoC-I on lipoproteins were determined by Scatchard analysis. The intrinsic affinity constant of antibody bound to apoC-I was estimated to be 5.49 X 10(10) M-1 whereas that of antibody binding to lipoproteins was 30 to 200 times less. Furthermore, ascites fluid from LRB 45 cell lines could immunoprecipitate serum lipoproteins. Thus, it is concluded that there is multiple binding of antibody to apoC-I on lipoproteins. This binding appears to increase the calculated affinity constant (avidity) for antibody-antigen interaction. 相似文献
9.
The kinetics of the reaction between human chorionic gonadotropin (hCG) and specific gonadotropin receptors in the rat testis were determined at 24 and 37 degrees, over a wide range of hormone concentrations. Hormone concentrations were corrected for the binding activity of the (-125I)hCG tracer preparations. Analysis of the experimental data was performed with an interactive nonlinear curve fitting program, based upon the second-order chemical kinetic differential equation. The mean values for the association rate constant (k1) were 4.7 x 10-7 M-1 min-1 at 24 degrees, and 11.0 x 10-7 M-1 min-1 at 37 degrees. At both temperatures, the values of kl were independent of hormone concentration. Initial dissociation rates were consistent with first order kinetics, with dissociation rate constant (k2) of 1.7 x 10 minus -3 and 4.6 x 10 minus -3 min minus -1 at 24 and 37 degrees, respectively. When studied over longer periods at 24 degrees, the dissociation process appeared to be multiexponential. The kinetics of degradation of (-125I)hCG and receptors were determined at both temperatures, and a mathematical model was developed by modification of the second-order chemical kinetic differential equation to take these factors into account. The application of such a model to hCG kinetic binding data demonstrated that reactant degradation had little significant effect on the derivation of the association rate constant (k1), but caused significant overestimation of the dissociation rate constant (k2) values derived from association experiments. The model was also applied by computer simulation to a theoretical analysis of the effects of degradation of free hormone and receptor sites upon kinetic and steadystate binding data. By this method, the initial velocities of hormone binding were shown to be less affected by degradation than the steady-state levels of hormone-receptor complex. Also, reactant degradation in simulated steady-state experiments caused an underestimate of the apparent equilibrium association constant, but had relatively less effect on the determination of binding site concentration. 相似文献
10.
A steady-state kinetic analysis was made of thiocyanate (SCN-) oxidation catalyzed by human peroxidase (SPO) isolated from parotid saliva. For comparative purposes, bovine lactoperoxidase (LPO) was also studied. Both enzymes followed the classical Theorell-Chance mechanism under the initial conditions [H2O2] less than 0.2mM, [SCN-] less than 10mM, and pH greater than 6.0. The pH-independent rate constants (k1) for the formation of compound I were estimated to be 8 X 10(6) M-1 s-1 (SD = 1, n = 18) for LPO and 5 X 10(6) M-1 s-1 (SD = 1, n = 11) for SPO. The pH-independent second-order rate constants (k4) for the oxidation of thiocyanate by compound I were estimated to be 5 X 10(6) M-1 s-1 (SD = 1, n = 18) for LPO and 9 X 10(6) M-1 s-1 (SD = 2, n = 11) for SPO. Both enzymes were inhibited by SCN- at pH less than 6. The pH-independent equilibrium constant (Ki) for the formation of the inhibited enzyme-SCN- complex was estimated to be 24 M-1 (SD = 12, n = 8) for LPO and 44 M-1 (SD = 4, n = 10) for SPO. An apparent pH dependence of the estimated values for k4 and Ki for both LPO and SPO was consistent with a mechanism based on assumptions that protonation of compound I was necessary for the SCN- peroxidation step, that a second protonation of compound I gave an inactive form, and that the inhibited enzyme-SCN- complex could be further protonated to give another inactive form.(ABSTRACT TRUNCATED AT 250 WORDS) 相似文献
11.
A simplified method has been developed for the determination of antibody-hapten association kinetics that permits the study of high affinity interactions with second order forward rate constants of the order of 10-7 to 10-8 M-1 sec-1. Use of tritiated haptens of high specific activity and antibodies of high affinity allows reactions to be run at initial hapten and antibody concentrations of the order of 10-9 to 10-10M, well below the level at which mixing becomes the rate-limiting step. Separation of antibody-bound from free hapten by the use of dextran-coated charcoal can be carried out with sufficient rapidity (2 sec) that the systems under investigation are not appreciably disturbed. With this technique, the association of 3-H-ouabain with rabbit ouabain-specific antibody was found to occur with a rate constant of 0.8 times 10-7 M-1 sec-1, similar to association rates of dye haptens with antibodies of substantially lower affinity. The ratio of this association rate constant to the independently determined dissociation rate constant was 5.4 times 10-9 M-1, in satisfactory agreement with a ko value of 3.5 times 10-9 M-1 determined by Sips analysis of data obtained under equilibrium conditions. This approach should be applicable to the direct kinetic assessment of numerous high affinity antibody-hapten systems of current interest. 相似文献
12.
Discoidal complexes of phosphatidylcholine (PC) . apolipoprotein A-I . cholesterol were prepared with egg PC, palmitoyloleoylPC, dipalmitoylPC, or dimyristoylPC, and were used as substrates of purified lecithin-cholesterol acyltransferase to investigate the effects of neutral salts on the enzymatic reaction. Sodium fluoride, chloride and bromide concentrations up to 1 M, did not affect the properties of the substrate particles, but caused marked and distinct changes in the activity of the enzyme with the various PC particles. The effects of salts were largely due to the anions, which followed the order of the lyotropic series in their inactivating capacity: F- less than Cl- less than Br- less than NO3- less than I- less than SCN-. Sodium salts (F-, Cl-, and Br-) produced a very large increase in the pH optimum of the enzymatic reaction (7.4 to at least 8.5) essentially obliterating the ionization of a functional group with pK of 8.1. The kinetics of the enzymatic reaction revealed major differences among the PC particles, and different responses of their kinetic parameters with increasing salt concentrations. The conclusions reached in this work are the following: (1) The relative reactivity of PC substrates, in discoidal particles, with lecithin-cholesterol acyltransferase depends strongly on the concentration and type of salts in the medium. (2) Anions (in lyotropic series) rather than cations affect the enzymatic reaction. (3) There are functional groups with pK of 8.1 which are affected markedly in their ionization behavior by anion binding. (4) The active site of lecithin-cholesterol acyltransferase and its interaction with anions are affected by the exact nature of the PC-apolipoprotein interface. 相似文献
13.
M Nakano 《Biochimica et biophysica acta》1982,707(1):115-120
Monovalent inorganic anions showed an unexpected effect on the inhibition of trehalase (alpha, alpha-trehalose glucohydrolase, EC 3.2.1.28) by SH inhibitors. This phenomeon (deinhibition) was caused by monovalent anions, Cl-, Br-, I- and SCN- . F- and ClO4- showed partial deinhibition. Deinhibition was not caused by NO2- and SO4-. The effectiveness of the "active anions' in causing deinhibition was highly dependent on the anion size. Trehalase in the presence of mercuric chloride was "activated' by Cl-, and the activation was saturable. From the results of Dixon plots for trehalase at different concentrations of the "activator' (deinhibitor) and a constant concentration of the substrate, it can be seen that the activator and the inhibitor competed with each other. Thus, it is suggested that the activator and the inhibitor share a common binding site or bind very near each other. The Ki value for mercuric chloride was increased with increasing concentration of NaCl. Therefore, it might be essential to remove the "active anions' in order to determine the inhibitory effect and the Ki value of trehalase for SH inhibitors. 相似文献
14.
Two lysine residues of bovine serum albumin reacted with 1-fluoro-2,4-dinitrobenzene with apparent second-order rate constants approx. 500-times greater than those observed in similar reactions with low-molecular-weight lysine derivatives. A series of dinitrophenyl (Dnp)-bovine serum albumins were prepared and their ability to bind univalent fragments of anti-Dnp antibody was measured by fluorescence-quenching titrations. Compared with the Dnp group of the free hapten, 6-N-Dnp-aminohexanoate, the majority of the protein-bound Dnp groups were unavailable to the antibody at pH8.0. When the same Dnp-albumins were titrated at pH3.0 the availability of the Dnp groups increased approx. 3-fold. Dnp-albumins were treated with pepsin at pH3.0 and Dnp-containing fragments isolated by chromatography on DE-52 DEAE-cellulose. Fluorescence-quenching titrations showed that the Dnp groups on the fragments behaved like the free hapten with respect to quenching efficiency, although with an increased dissociation constant. The association between the Dnp-albumins and the antibody was measured also by difference-spectral titrations at high protein concentrations. Antibody binding was increased under these conditions, but the Dnp group of mono-Dnp-albumin remained unavailable to antibody. We propose that the reactive lysine residues are located in clefts between the globular sub-domains of the single polypeptide chain. Dnp groups attached to these lysine residues are fully exposed to the solvent, but binding of the macromolecular probe, anti-Dnp antibody, is sterically hindered by the adjacent surface of the albumin molecule. 相似文献
15.
E L?vaas 《Free radical biology & medicine》1992,13(3):187-195
The lactoperoxidase-catalyzed oxidation of glutathione (GSH) and thiocyanate (SCN-) was studied. Oxidation of SCN- was recorded by ultraviolet spectroscopy and by electron spin resonance (ESR). Consumption of GSH was measured by amperometric titration. One or two moles of GSH was oxidized per mole of H2O2 added, depending on the reaction conditions. Omission of SCN- prevented the oxidation of GSH. The oxidation of GSH required only catalytic amounts of SCN-, which was therefore recycled. Iodide (I-) could replace SCN-, while chloride or bromide were ineffective. The apparent Michaelis constant for SCN- was 17 microM. Oxidation of SCN- gave rise to two reactive intermediates, one stable and one unstable. The stable intermediate (-OSC. = N-(?)) decayed by a second-order reaction with a rate constant of 1.1 M-1 s-1. The decay of the unstable radical was very fast. The data (a) explain the short- and long-term antibacterial effects of lactoperoxidase-halide-H2O2 system, (b) point to possible deleterious effects due to glutathione depletion, (c) are of relevance for free radical diseases involving sulphur-centered free radicals, and (d) support previous observations on lipid peroxidation/halogenation in biological membranes, liposomes, and unsaturated fatty acids. 相似文献
16.
The reaction of human myeloperoxidase with its product, hypochlorous acid was investigated using both rapid-scan spectrophotometry and the stopped-flow technique. In the reaction of myeloperoxidase with hypochlorous acid a primary compound is found with properties similar to that of compound I and which is converted into compound II. The primary reaction is strongly pH-dependent. At pH 7.2 the reaction is too fast to be measured but at higher pH values it is possible to determine the apparent second-order rate constant. Its value decreases to about 2 x 10(7) M-1.s-1 at pH 8.3 and to 2.3 (+/- 0.4) x 10(6) M-1.s-1 at pH 9.2, respectively. The dissociation constant for the formation of the primary compound is 25.7 (+/- 15.3) microM at pH 9.2 and about 2.5 microM at pH 8.3. The apparent second-order rate constant for the formation of compound II is hardly affected by pH and varies between 2 to 5 x 10(4) M-1.s-1 at pH 10.2 and pH 8.3, respectively. Reaction of myeloperoxidase with hypochlorous acid also resulted in irreversible partial bleaching of the chromophore. Chloride, which is a substrate of the enzyme not only protects myeloperoxidase against bleaching by hypochlorous acid but also competitively inhibits the binding of hypochlorous acid to myeloperoxidase, a process which also has been observed in the reaction with hydrogen peroxide. It is concluded that hypochlorous acid binds at the heme iron to form compound I. 相似文献
17.
We have measured the kinetic properties of the Escherichia coli cAMP receptor protein (CAP) and lac repressor interacting with lac promoter restriction fragments. Under our reaction conditions (10 mM-Tris X HCl (pH 8.0 at 21 degrees C), 1 mM-EDTA, 10 microM-cAMP, 50 micrograms bovine serum albumin/ml, 5% glycerol), the association of CAP is at least a two-step process, with an initial, unstable complex formed with rate constant kappa a = 5(+/- 2.5) X 10(7) M-1 s-1. Subsequent formation of a stable complex occurs with an apparent bimolecular rate constant kappa a = 6.7 X 10(6) M-1 s-1. At low total DNA concentration, the dissociation rate constant for the specific CAP-DNA complex is 1.2 X 10(-4) s-1. The ratio of formation and dissociation rate constants yields an estimate of the equilibrium constant, Keq = 5 X 10(10) M-1, in good agreement with static results. We observed that the dissociation rate constant of both CAP-DNA and repressor-DNA complexes is increased by adding non-specific "catalytic" DNA to the reaction mixture. CAP dissociation by the concentration-dependent pathway is second-order in added non-specific DNA, consistent with either the simultaneous or the sequential participation of two DNA molecules in the reaction mechanism. The results imply a role for distal DNA in assembly-disassembly of specific CAP-DNA complexes, and are consistent with a model in which the subunits in the CAP dimer separate in the assembly-disassembly process. The dissociation of lac repressor-operator complexes was found to be DNA concentration-dependent as well, although in contrast to CAP, the reaction is first-order in catalytic DNA. Added excess operator-rich DNA gave more rapid dissociation than equivalent concentrations of non-specific DNA, indicating that the sequence content of the competing DNA influences the rate of repressor dissociation. The simplest interpretation of these observations is that lac repressor can be transferred directly from one DNA molecule to another. A comparison of the translocation rates calculated for direct transfer with those predicted by the one-dimensional sliding model indicates that direct transfer may play a role in the binding site search of lac repressor. 相似文献
18.
Manganese peroxidase from the lignin-degrading basidiomycete Phanerochaete chrysosporium. Transient state kinetics and reaction mechanism 总被引:15,自引:0,他引:15
H Wariishi H B Dunford I D MacDonald M H Gold 《The Journal of biological chemistry》1989,264(6):3335-3340
Stopped-flow techniques were used to investigate the kinetics of the formation of manganese peroxidase compound I (MnPI) and of the reactions of MnPI and manganese peroxidase compound II (MnPII) with p-cresol and MnII. All of the rate data were obtained from single turnover experiments under pseudo-first order conditions. In the presence of H2O2 the formation of MnPI is independent of pH over the range 3.12-8.29 with a second-order rate constant of (2.0 +/- 0.1) x 10(6) M-1 s-1. The activation energy for MnPI formation is 20 kJ mol-1. MnPI formation also occurs with organic peroxides such as peracetic acid, m-chloroperoxybenzoic acid, and p-nitroperoxybenzoic acid with second-order rate constants of 9.7 x 10(5), 9.5 x 10(4), and 5.9 x 10(4) M-1 s-1, respectively. The reactions of MnPI and MnPII with p-cresol strictly obeyed second-order kinetics. The second-order rate constant for the reaction of MnPII with p-cresol is extremely low, (9.5 +/- 0.5) M-1 s-1. Kinetic analysis of the reaction of MnII with MnPI and MnPII showed a binding interaction with the oxidized enzymes which led to saturation kinetics. The first-order dissociation rate constants for the reaction of MnII with MnPI and MnPII are (0.7 +/- 0.1) and (0.14 +/- 0.01) s-1, respectively, when the reaction is conducted in lactate buffer. Rate constants are considerably lower when the reactions are conducted in succinate buffer. Single turnover experiments confirmed that MnII serves as an obligatory substrate for MnPII and that both oxidized forms of the enzyme form productive complexes with MnII. Finally, these results suggest the alpha-hydroxy acids such as lactate facilitate the dissociation of MnIII from the enzyme. 相似文献
19.
The binding of aqueous anions (ClO4-, SCN-, I-, and NO3-) to lipid bilayer membranes composed of 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) was investigated using deuterium (2H) and phosphorus-31 (31P) nuclear magnetic resonance (NMR) spectroscopy. The ability of these anions to influence the 2H NMR quadrupole splittings of POPC, specifically labeled at the alpha or beta position of the choline head group, increased in the order NO3- much less than I- less than SCN- less than ClO4-. In the presence of these chaotropic anions, the quadrupole splitting increased for alpha-deuterated POPC and decreased for beta-deuterated POPC, indicating a progressive accumulation of negative charge at the membrane surface. Calibration of the 2H NMR quadrupole splittings with the amount of membrane-bound anion permitted binding isotherms to be generated for perchlorate, thiocyanate, and iodide, up to concentrations of 100 mM. The binding isotherms were analyzed by considering electrostatic contributions, according to the Gouy-Chapman theory, as well as chemical equilibrium contributions. For neutral POPC membranes, we obtained ion association constants of 32, 80, and 115 M-1 for iodide, thiocyanate, and perchlorate, respectively. These values increase in the order expected for a Hofmeister series of anions. We conclude that the factor determining whether a particular anion will bind to lipid bilayers is the ease with which that anion loses its hydration shell.(ABSTRACT TRUNCATED AT 250 WORDS) 相似文献
20.
gamma-Aminobutyric acid (GABA) receptor-mediated 36chloride (36Cl-) uptake was measured in synaptoneurosomes from rat brain. GABA and GABA agonists stimulated 36Cl- uptake in a concentration-dependent manner with the following order of potency: Muscimol greater than GABA greater than piperidine-4-sulfonic acid (P4S)greater than 4,5,6,7-tetrahydroisoxazolo-[5,4-c]pyridin-3-ol (THIP) = 3-aminopropanesulfonic acid (3APS) much greater than taurine. Both P4S and 3APS behaved as partial agonists, while the GABAB agonist, baclofen, was ineffective. The response to muscimol was inhibited by bicuculline and picrotoxin in a mixed competitive/non-competitive manner. Other inhibitors of GABA receptor-opened channels or non-neuronal anion channels such as penicillin, picrate, furosemide and disulfonic acid stilbenes also inhibited the response to muscimol. A regional variation in muscimol-stimulated 36Cl- uptake was observed; the largest responses were observed in the cerebral cortex, cerebellum and hippocampus, moderate responses were obtained in the striatum and hypothalamus and the smallest response was observed in the pons-medulla. GABA receptor-mediated 36Cl- uptake was also dependent on the anion present in the media. The muscimol response varied in media containing the following anions: Br- greater than Cl- greater than or equal to NO3- greater than I- greater than or equal to SCN- much greater than C3H5OO- greater than or equal to ClO4- greater than F-, consistent with the relative anion permeability through GABA receptor-gated anion channels and the enhancement of convulsant binding to the GABA receptor-gated Cl- channel. 相似文献