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1.
The kinetics of ethenoadenosine triphosphate (?ATP) as the phosphate donor in the phosphoryl transfer reaction of hexokinase were examined to obtain the Km′s, V's, and Kα's for the nucleotide and sugar. Dissociation constants for eATP and ?ADP with hexokinase were obtained from fluorometric measurements and compared with similar constants obtained kinetically. Other selected nucleoside triphosphates were used as phosphate donors in the hexokinase reaction and their kinetic constants were obtained. Reactions were also performed using two nucleotides simultaneously as phosphorylating substrates for the hexokinase reaction in an attempt to find the individual dissociation constants, Km′s and Ki′s. These were compared with the Km′s obtained from using the nucleotides separately in the hexokinase reaction. From these kinetic and fluorescence binding studies, evidence is presented supporting the postulate that the Km′s are primarily dissociation constants in a random bi-bi mechanism. Analysis of the Km values provides additional evidence to support the importance of the amino group in position 6 on the purine ring as a hydrogen-bond acceptor during binding. It was found that ?CTP was a much better hexokinase substrate than CTP. These observations suggest that the V for this reaction is highly dependent upon the size of the nucleotide. 相似文献
2.
1. The Teorell, Meyer-Sievers theory characterizes the electrochemical behavior of membranes by their selectivity constant "Ap" which is derived conventionally from concentration potential measurements at various concentration levels. The selectivity constant may, however, be derived also from entirely independent, different experimental data, namely base exchange studies. The constants arrived at in this second way are designated as "Ab." The selectivity constants derived by these two methods must be in reasonable, at least semiquantitative agreement if the basic assumptions of the theory are correct. 2. The selectivity constants Ap and Ab were determined for eleven different sets of membranes of different electrochemical activity and of different (8.2 to 80 volume per cent) water content. 3. The potentiometric selectivity constants Ap are in most cases several orders of magnitude greater than the corresponding Ab values. With membranes of great porosity and high electrochemical activity the Ab values approach at least in order of magnitude the Ap values. 4. It is concluded that the unexpectedly large discrepancy between the Ap and Ab values is due to some inherent weakness of the Teorell, Meyer-Sievers theory, most likely to its neglect of any structural factors. 相似文献
3.
Most of the important types of interactions that occur in cells can be characterized as binding-diffusion type processes, and can be quantified by kinetic rate constants such as diffusion coefficients (D) and binding rate constants (kon and koff). Confocal FRAP is a potentially important tool for the quantitative analysis of intracellular binding-diffusion kinetics, but how to dependably extract accurate kinetic constants from such analyses is still an open question. To this end, in this study, we developed what we believe is a new analytical model for confocal FRAP-based measurements of intracellular binding-diffusion processes, based on a closed-form equation of the FRAP formula for a spot photobleach geometry. This approach incorporates a binding diffusion model that allows for diffusion of both the unbound and bound species, and also compensates for binding diffusion that occurs during photobleaching, a critical consideration in confocal FRAP analysis. In addition, to address the problem of parametric multiplicity, we propose a scheme to reduce the number of fitting parameters in the effective diffusion subregime when D's for the bound and unbound species are known. We validate this method by measuring kinetic rate constants for the CAAX-mediated binding of Ras to membranes of the endoplasmic reticulum, obtaining binding constants of kon ∼ 255/s and koff ∼ 31/s. 相似文献
4.
K E Neet 《Biophysical chemistry》1974,2(2):102-115
Conformationally-linked dissociation equilibria of dimeric proteins have been examined to determine how experimentally obtainable parameters, such as the apparent dissociation constant, kD, and the apparent conformational transition constant, Kconf, are related to intrinsic subunit interaction constants, KA or KB, and intrinsic isomerization constants, K1 or k2. Limiting models are considered in which either the conformational change occurs before dissociation or in which the dissociation occurs before the conformational change, as well as a general model including both possibilities. Models are also considered in which three conformations are allowed or in which four subunits (tetramers) are involved. Simulated data for the dimer equilibria are presented to show how variation of protein concentration and variation of certain constants affect the proportion of various molecular species, the weight-average molecular weight, and the overall extent of conformational change. Methods are suggested to distinguish between the different limiting cases based upon the dependence of KD and/or Kconf on protein concentration, perturbant concentration, and temperature. It is concluded that methods used to calculate self-dissociation constants oligomeric proteins include linked isomerization reactions such that the equilibrium constant obtained should not be considered as a true subunit interaction term. Indeed, dissociation can occur under the influence of a perturbant with no change in the intrinsic affinity of the subunits but with the sole effect of the perturbant being on a linked conformational change. Additional experiments on the thermodynamics of the conformational change are required to determine the actual relationship. Depending on the complexity of the equilibria involved and the relative value of the equilibrium constants, the extent of the conformational change can vary directly with, vary inversely with, or he independent of the total protein concentration. Even when intrinsic subunit affinities are not affected by the perturbant, the extent of conformational change can vary with protein concentration. Interpretation of data from proteins which may be involved in conformationally-linked dissociation reactions, therefore, must be made with caution. 相似文献
5.
6.
Charge-pulse relaxation experiments of valinomycin-mediated Rb+ transport have been carried out in order to study the influence of membrane structure on carrier kinetics. From the experimental data the rate constants of association (kR) and dissociation (kD) of the ion-carrier complex as well as the rate constants of translocation of the complex (kMS) and of the free carrier (kS) could be obtained. The composition of the planar bilayer membrane was varied in a wide range. In a first series of experiments, membranes made from glycerolmonooleate dissolved in different n-alkanes (n-decane to n-hexadecane), as well as solvent-free membranes made from the same lipid by the Montal-Mueller technique were studied. The translocation rate constants kS and kMS were found to differ by less than a factor of two in the membranes of different solvent content. Much larger changes of the rate constants were observed if the structure of the fatty acid residue was varied. For instance, an increase in the number of double bonds in the C20 fatty acid from one to four resulted in an increase of kS by a factor of seven and in an increase of kMS by a factor of twenty-four. The stability constant K = kR/kD of the ion-carrier complex as well as the translocation rate constants kS and kMS were found to depend strongly on the nature of the polar headgroup of the lipid. The incorporation of cholesterol into glycerolmonooleate membranes reduced kR, kMS and kS up to seven-fold. 相似文献
7.
Actin is an abundant protein that constitutes a main component of the eukaryotic cytoskeleton. Its polymerization and depolymerization are regulated by a variety of actin-binding proteins. Their functions range from nucleation of actin polymerization to sequestering G-actin in 1∶1 complexes. The kinetics of forming these complexes, with rate constants varying at least three orders of magnitude, is critical to the distinct regulatory functions. Previously we have developed a transient-complex theory for computing protein association mechanisms and association rate constants. The transient complex refers to an intermediate in which the two associating proteins have near-native separation and relative orientation but have yet to form short-range specific interactions of the native complex. The association rate constant is predicted as k
a = k
a0
, where k
a0 is the basal rate constant for reaching the transient complex by free diffusion, and the Boltzmann factor captures the bias of long-range electrostatic interactions. Here we applied the transient-complex theory to study the association kinetics of seven actin-binding proteins with G-actin. These proteins exhibit three classes of association mechanisms, due to their different molecular shapes and flexibility. The 1000-fold k
a variations among them can mostly be attributed to disparate electrostatic contributions. The basal rate constants also showed variations, resulting from the different shapes and sizes of the interfaces formed by the seven actin-binding proteins with G-actin. This study demonstrates the various ways that actin-binding proteins use physical properties to tune their association mechanisms and rate constants to suit distinct regulatory functions. 相似文献
8.
Methods are described for extracting the rate constants, governing the transitions between states, for a generalized, linear three discrete state model for the Na conductance,g Na. It is shown that the problem cannot be solved given only the time course ofg Na during some test potential step with fixed initial conditions. However, if the effects of any two durations of some conditioning potential step on the peakg Na during the test step are taken into account, and the physically required assumption that the steady state is an equilibrium state is made, then the rate constants for both conditioning and test potentials can be solved for simultaneously. These methods were applied to thev 5 model of Goldman (5) in which theg Na(t) was described in terms of a generalized linear second order variable. After extensive computations involving some 20,000 combinations of experimentally determined input values, it was not found possible to extract a set of rate constants that were all real and positive as is required. It was concluded that if the transitions between states are first order and there are no subunit interactions, then the Na gating unit displays more than three states. 相似文献
9.
Estimating Kinetic Parameters when the Amount of Enzyme Added to an Assay Is Not a Controlled Variable: NITROGENASE ACTIVITY OF INTACT LEGUMES
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Davis LC 《Plant physiology》1980,66(1):126-129
Reliable estimates of Michaelis constants (Km) and inhibitor constants may be obtained, in the absence of control over the amount of enzyme being added to any assay system, provided the following constraints are met. Michaelis-Menten kinetics are obeyed. Two rate measurements must be made with the same sample of enzyme: at low and high substrate concentration for determining Km or minus and plus an inhibitor for determining inhibitor constants. The Michaelis constant may be calculated from the equation [Formula: see text] Inhibitor constants are derived graphically from Lineweaver-Burk or Dixon plots, once the Km has been calculated. The above technique has been applied to study of the acetylene-reducing ability of intact legume plants. The apparent Km for acetylene reduction by nitrogenase in legume nodules is ~1/100 atmosphere in the absence of nitrogen and ~1/40 atmosphere in its presence. 相似文献
10.
We report the synthesis of the Schiff base ligands, 4-[(4-bromo-phenylimino)-methyl]-benzene-1,2,3-triol (A1), 4-[(3,5-di-tert-butyl-4-hydroxy-phenylimino)-methyl]-benzene-1,2,3-triol (A2), 3-(p-tolylimino-methyl)-benzene-1,2-diol (A3), 3-[(4-bromo-phenylimino)-methyl]-benzene-1,2-diol (A4), and 4-[(3,5-di-tert-butyl-4-hydroxy-phenylimino)-methyl]-benzene-1,3-diol (A5), and their Cd(II) and Cu(II) metal complexes, stability constants and potentiometric studies. The structure of the ligands and their complexes was investigated using elemental analysis, FT-IR, UV-Vis, 1H and 13C NMR, mass spectra, magnetic susceptibility and conductance measurements. In the complexes, all the ligands behave as bidentate ligands, the oxygen in the ortho position and azomethine nitrogen atoms of the ligands coordinate to the metal ions. The keto-enol tautomeric forms of the Schiff base ligands A1-A5 have been investigated in polar and non-polar organic solvents. Antimicrobial activity of the ligands and metal complexes were tested using the disc diffusion method and the strains Bacillus megaterium and Candida tropicalis.Protonation constants of the triol and diol Schiff bases and stability constants of their Cu2+ and Cd2+ complexes were determined by potentiometric titration method in 50% DMSO-water media at 25.00 ± 0.02 °C under nitrogen atmosphere and ionic strength of 0.1 M sodium perchlorate. It has been observed that all the Schiff base ligands titrated here have two protonation constants. The variation of protonation constant of these compounds was interpreted on the basis of structural effects associated with the substituents. The divalent metal ions of Cu2+ and Cd2+ form stable 1:2 complexes with Schiff bases.The Schiff base complexes of cadmium inhibit the intense chemiluminescence reaction in dimethylsulfoxide (DMSO) solution between luminol and dioxygen in the presence of a strong base. This effect is significantly correlated with the stability constants KCdL of the complexes and the protonation constants KOH of the ligands; it also has a nonsignificant association with antibacterial activity. 相似文献
11.
Xinyu Li Jia Liu Shanshan Liang Kuiwen Guan Lijia An Xuefei Wu Shao Li Changsen Sun 《Cell biochemistry and biophysics》2013,67(3):1409-1419
Near-infrared laser provides a novel nerve stimulation modality to regulate the cell functions. Understanding its physiological effect is a prerequisite for clinic laser therapy applications. Here, the whole-cell sodium (Na) channel kinetics of neuron cell was employed to determine the temporal roles of infrared laser. The Na currents were elicited by electrical pulses that were synchronized at the rising and falling edges of the 980 nm laser pulses, respectively, to investigate the different infrared effect on cell functions. The time constants of activation (τ m) and inactivation (τ h) kinetics were extracted from fitting of the Na current (m3h) according to the Hodgkin–Huxley (HH) model. By comparing the time constants without and with the laser irradiation, we obtained that laser pulses changed the Na current kinetics by accelerating τ h-phase and slowing down τ m-phase at the beginning of the laser pulse, whereas both phases were accelerated at the end of the pulse. After relating the ratios of the time constants to the temperature characteristics of Na channel by Q 10, we found that the accelerating in Na current kinetics could be related to the average temperature of extracellular solution in the corresponding time span by choosing Q 10 = 2.6. The results of this study demonstrated that there was a positive correlation between the acceleration of the Na current kinetics and increases in temperature of the extracellular solution. 相似文献
12.
13.
Elin Säwén 《Carbohydrate research》2010,345(8):984-1319
The synthesis of two novel carbasugar analogues of α-l-iduronic acid is described in which the ring-oxygen is replaced by a methylene group. In analogy with the conformational equilibrium described for α-l-IdopA, the conformation of the carbasugars was investigated by 1H and 13C NMR spectroscopy. Hadamard transform NMR experiments were utilised for rapid acquisition of 1H,13C-HSQC spectra and efficient measurements of heteronuclear long-range coupling constants. Analysis of 1H NMR chemical shifts and JH,H coupling constants extracted by a total-lineshape fitting procedure in conjunction with JH,C coupling constants obtained by three different 2D NMR experiments, viz., 1H,13C-HSQC-HECADE, J-HMBC and IPAP-HSQC-TOCSY-HT, as well as effective proton-proton distances from 1D 1H,1H T-ROE and NOE experiments showed that the conformational equilibrium 4C1?2S5a?1C4 is shifted towards 4C1 as the predominant or exclusive conformation. These carbasugar bioisosteres of α-l-iduronic acid do not as monomers show the inherent flexibility that is anticipated to be necessary for biological activity. 相似文献
14.
Robert S. Slaughter Raymond G. Fenwick Eugene M. Barnes 《Archives of biochemistry and biophysics》1981,211(1):494-499
The transport of thymidine and hypoxanthine was investigated in mutant Chinese hamster lung fibroblasts deficient in both thymidine kinase and hypoxanthine-guanine phosphoribosyltransferase. Kinetic data from rapid uptake experiments (0.5–4.5 s) indicate that thymidine is transported by a monophasic saturable system (Km = 0.29 mM, V = 6.7 nmol/min · mg) which is competitively inhibited by hypoxanthine (Ki = 3.3 mM). The cells displayed a single transport system for hypoxanthine (Km = 2.0 mM, V = 8.9 nmol/min · mg) that is inhibited competitively by thymidine (Ki = 0.43 mM). Both hypoxanthine and thymidine entry were noncompetively inhibited by nitrobenzylthioinosine, but thymidine transport was more sensitive. A kinetic model in which hypoxanthine and thymidine share a common transporter can account for the competitive inhibition and the observation that the inhibition constants are similar to the Michaelis constants. 相似文献
15.
N. L. Zakharchenko E. A. Ermakova Yu. F. Zuev 《Russian Journal of Bioorganic Chemistry》2008,34(3):364-368
The hydrolysis reaction of N α-benzoyl-L-arginine ethyl ester catalyzed by trypsin from pig pancreas was comparatively studied in an aqueous buffer solution and in the system of reversed micelles of Aerosol OT in octane (pH 8.5) to determine the mechanisms of influence of the enzyme microenvironment on the rate constants of the elementary stages of the enzymatic reaction. The temperature dependences of the catalytic constant k cat and the rate constant of the second order k cat/K m (s, catalysis efficiency) allowed the determination of the rate constants and the activation energy of elementary stages of the enzymatic reaction. It was revealed that a decrease in the efficiency of catalytic action of trypsin in reverse micelles in comparison with an aqueous solution is first of all determined by a decrease in the rate constant of formation of the enzyme-substrate complex k 1. Possible mechanisms of the effect of the microenvironment on the elementary stages of catalytic action of the enzyme are discussed. 相似文献
16.
Matthew B. Russell Christopher W. Woodall Shawn Fraver Anthony W. D’Amato Grant M. Domke Kenneth E. Skog 《Ecosystems》2014,17(5):765-777
A key component in describing forest carbon (C) dynamics is the change in downed dead wood biomass through time. Specifically, there is a dearth of information regarding the residence time of downed woody debris (DWD), which may be reflected in the diversity of wood (for example, species, size, and stage of decay) and site attributes (for example, climate) across the study region of eastern US forests. The empirical assessment of DWD rate of decay and residence time is complicated by the decay process itself, as decomposing logs undergo not only a reduction in wood density over time but also reductions in biomass, shape, and size. Using DWD repeated measurements coupled with models to estimate durations in various stages of decay, estimates of DWD half-life (T HALF), residence time (T RES), and decay rate (k constants) were developed for 36 tree species common to eastern US forests. Results indicate that estimates for T HALF averaged 18 and 10 years for conifers and hardwoods, respectively. Species that exhibited shorter T HALF tended to display a shorter T RES and larger k constants. Averages of T RES ranged from 57 to 124 years for conifers and from 46 to 71 years for hardwoods, depending on the species and methodology for estimating DWD decomposition considered. Decay rate constants (k) increased with increasing temperature of climate zones and ranged from 0.024 to 0.040 for conifers and from 0.043 to 0.064 for hardwoods. These estimates could be incorporated into dynamic global vegetation models to elucidate the role of DWD in forest C dynamics. 相似文献
17.
The binding constants, K1 and K2, and the number of Ca2+ ions in each of the two high affinity sites of Ca2+-regenerated bacteriorhodopsin (bR) are determined potentiometrically at different pH values in the range of pH 3.5-4.5 by using the Scatchard plot method. From the pH dependence of K1 and K2, it was found that two hydrogen ions are released for each Ca2+ bound to each of the two high affinity sites. Furthermore, we have measured by a direct spectroscopic method the association constant, Ks, for the binding of Ca2+ to deionized bR, which is responsible for producing the blue to purple color change. Comparing the value of Ks and its pH dependence with those of K1 and K2 showed that the site corresponding to Ks is to be identified with that of K2. This is in agreement with the conclusion reached previously, using a different approach, which showed that it is the second Ca2+ that causes the blue to purple color change.
Our studies also show that in addition to the two distinct high affinity sites, there are about four to six sites with lower binding constants. These are attributed to the nonspecific binding in bR.
相似文献18.
Joerg Koehler Markus Beck Erlach Edson Crusca Jr. Werner Kremer Claudia E. Munte Alexander Meier Hans Robert Kalbitzer 《Journal of biomolecular NMR》2014,60(1):45-50
The pressure dependence of the one-bond indirect spin–spin coupling constants 1 J N–H was studied in the protected tetrapeptides Ac-Gly-Gly-Xxx-Ala-NH2 (with Xxx being one of the 20 proteinogenic amino acids). The response of the 1 J N–H coupling constants is amino acid type specific, with an average increase of its magnitude by 0.6 Hz at 200 MPa. The variance of the pressure response is rather large, the largest pressure effect is observed for asparagine where the coupling constant becomes more negative by ?2.9 Hz at 200 MPa. The size of the J-coupling constant at high pressure is positively correlated with its low pressure value and the β-propensity, and negatively correlated with the amide proton shift and the first order nitrogen pressure coefficient and the electrostatic solvation free energy. 相似文献
19.
The formation constants of the proton and calcium complexes of PGE1 and PGF1β have been measured in aqueous solution at 20 ± 1°. The proton complexes are shown to be effectively ionized at pH 7, log KHL values being 5.12 and 5.46, respectively. The calcium complexes, studied radiometrically using 45Ca by ion exchange equilibria, have formation constants (KCaPG) of 37 and 9 (approx.), respectively. “Model” ligands studied in developing the technique were 3-hydroxybutyric acid (KCa, 3.1) and 4-hydroxybutyric acid (KCa, 5.7). 相似文献
20.
Electric field pulses induce a substantial increase of the light scattering intensity of double-helical DNA. The relative change of light scattering and also the reciprocal relaxation time constants under electric field pulses increase with increasing nucleotide concentration. These observations, together with a large difference between dichroism orientation time constants and light scattering time constants under electric field pulses, demonstrate that the main part of the light scattering effect is due not to field-induced orientation but to interactions between DNA helices. From the concentration dependence of the light scattering time constants we obtain, according to an isodesmic reaction model, association rate constants in the range 3 × 1010 M?1 helices s?1 for DNA with approx. 300 base-pairs. These values are at the limit of a diffusion-controlled DNA association and do not show any dependence upon the field strength. The dissociation rate constants kd decrease strongly with increasing field strength E and thus demonstrate that the interactions between the helices are induced by the electric field. This conclusion is consistent with independent measurements which do not reveal any DNA association at zero field strength. The observed linear relation between log(kd) and E2 suggests a field-induced reaction driven by dipole changes. According to this interpretation the change of dipole moment should be in the range of approx. 1400 debye. The dissociation rates for DNA helices with approx. 300 to approx. 800 base-pairs strongly increase with increasing sail concentration (measured in the range 1–5 mM ionic strength), whereas the association rate constants remain virtually unchanged. Measurements of the linear dichroism in the same range of DNA chain length demonstrate that for long field pulses of e.g., 40 μs, the amplitude approaches a maximum value and then decreases. The dichroism relaxation curves observed after long field pulses exhibit a component with a positive dichroism and an increased decay time. These observations suggest the formation of a DNA aggregate with an unusual arrangement of the bases. 相似文献