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1.
Hélène Bador Renée Morelis Pierre Louisot 《Biochimica et Biophysica Acta (BBA)/General Subjects》1984,800(1):75-86
Temperature dependence of asialomucin-sialyltransferase () N-acetylneuraminyltransferase, EC 2.4.99.1) activity is investigated. Discontinuities in Arrhenius plots are observed, whether the enzyme is membrane-associated or solubilized. These discontinuities cannot be firmly correlated with the phase-transition temperatures of either endogenous or exogenous phospholipids. Arrhenius plots of the kinetic parameters also exhibit sharp discontinuities, so that it is concluded that a significant change in Km and Vmax values occurs with varying temperature. Our results suggest that the biphasic behavior of Arrhenius plots may be attributed to the temperature dependence of the kinetic parameters for both membrane-associated and solubilized sialyltransferase activities. 相似文献
2.
The Michaelis constant and maximal velocity of alpha-amylase-free amylo-glucosidase decrease with increasing periodate oxidation of amylose. These kinetic features have been explained on the basis of competitive inhibition by the oxidised non-reducing end of the (1→4)-α-d-glucan chain with the active centres of the enzyme. A kinetic model is proposed to demonstrate this special kind of inhibition where the concentration of inhibitor is directly proportional to the substrate concentration. The experimental data fitted this model, and the plots of 1/Km and 1/V against the ratio or oxidised/unoxidised non-reducing end-groups were straight lines. 相似文献
3.
A study has been made of the kinetics of condensation reactions in phosphotriester synthesis of oligonucleotides on polystyrene copolymer resin support, using monomers, dimers, and trimers as the coupling units. The condensations follow pseudo first order kinetics, with a rate constant that decreases from monomers to dimers to trimers. Arrhenius plots indicate a nearly constant activation energy of around 10.4 kcal mol-1. Extrapolation to find the Arrhenius preexponential factor, A, is inaccurate because of the limited temperature range studied. But if the assumption is made that Ea is the same for monomer, dimer, and trimer reactions, then the A values are inversely proportional to the surface areas of the coupling units, reinforcing the interpretation of A as a collision efficiency factor. 相似文献
4.
E T Rakitzis 《The Biochemical journal》1989,263(3):855-859
A kinetic analysis is presented of reactions of protein modification, and/or of modification-induced enzyme inactivation, which can formally be described by a single exponential function, or by a summation of two exponential functions, of reaction time plus a constant term. The reaction schemes compatible with the kinetic formalism of these cases are given, and a simple kinetic criterion is described whereby the identification of one of these cases, strong negative protein modification co-operativity, may be carried out. The treatment outlined in this paper is applied to a case from the literature, the inactivation of glyceraldehyde-3-phosphate dehydrogenase by butane-2,3-dione [Asriyants, Benkevich & Nagradova (1983) Biokhimiya (Engl. Transl.) 48, 164-171]. 相似文献
5.
In contrast with the ease of observing heterotropic effects in allosteric enzymes of low co-operativity, the detection of homotropic effects is often difficult. As a consequence, erroneous conclusions about the uncoupling of homotropic and heterotropic effects can result unless sensitive techniques are used for analyzing the kinetic data. Simulations of experiments as well as actual measurements on the allosteric enzyme, aspartate transcarbamoylase, of Escherichia coli and some of its modified forms, were performed in attempts to develop stringent diagnostic procedures for the detection of homotropic effects in enzymes of low co-operativity. The analyses show that direct saturation plots (velocity versus substrate concentration), double reciprocal plots, and Hill plots yield misleading results in that the co-operativity known to be present is not observed. In contrast, Eadie plots (velocity/substrate concentration versus velocity) are much more sensitive in revealing homotropic effects. Since the observed co-operativity depends on both the allosteric equilibrium constant, L, and the number of active sites, n, simulations were performed on the effect of those parameters. The maxima in the Eadie plots increased as L was lowered and conversely the maxima decreased as n was reduced. These changes were confirmed with a mutant aspartate transcarbamoylase which had the same specific activity as the wild-type enzyme and a lower value of L, and also with a hybrid enzyme containing fewer active sites and the same L value. Analogous experiments on nitrated aspartate transcarbamoylase derivatives of decreasing activity showed that Eadie plots were of value in distinguishing between the changes in L and n values resulting from the inactivation. Data from the literature were analyzed in the form of Eadie plots and in all cases homotropic effects were readily detectable for aspartate transcarbamoylase derivatives previously claimed to be devoid of co-operativity. 相似文献
6.
中梁山岩溶槽谷区不同土地利用方式坡地产流规律 总被引:4,自引:1,他引:3
坡地产流是造成岩溶区水土流失的主要驱动力,研究典型岩溶槽谷区坡地产流规律,对岩溶区防治水土流失、合理利用地下水资源具有重要理论意义。在重庆市中梁山龙凤和龙车槽谷选取不同土地利用方式的4个标准径流小区,对降水、地表径流、壤中流、裂隙流和土壤含水率进行了同步监测,探讨了坡地产流特征。结果表明:(1)4个不同土地利用方式的径流小区,坡地总产流量从大到小依次为:耕地(3696.9L)果园地(3657.2L)竹林地(2922.9L)林地(2211.1L),总径流系数(3.1%—5.2%)远低于非岩溶区(约20%);(2)4个径流小区的产流形式主要为地表径流,壤中流和裂隙流产生滞后于地表径流;(3)降水因子、前期土壤含水率共同影响地表径流,但降水因子对地表径流的影响远大于前期土壤含水率。降水因子中,15min最大雨强是影响耕地、果园地的地表径流的主要因素,降水量是影响林地、竹林地的地表径流的主要因素;前期土壤含水率对耕地、林地、果园地地表径流影响较大,对竹林地地表径流影响较小。 相似文献
7.
The steady-state kinetics of DT diaphorase [NAD(P)H dehydrogenase (quinone): EC 1.6.99.2]from rat liver has been studied using 2,6-dichloroindophenol as electron acceptor and NADH as electron donor. The υ vs [S]curves revealed intermediary plateau and “trough” regions when NADH and dichloroindophenol were the varying substrates. It has been demonstrated by statistical analysis that equations involving fourth-power terms of the substrate concentrations are able to describe the “trough.” These equations have been previously shown in the literature to be capable of describing intermediary plateau regions also. Changes in protein concentration did not affect these curves substantially, whereas changes in pH caused shifts in the positions of their intermediary plateau regions. Increase of the ionic strength by addition of 0.25–0.5 m KCl changed the intermediary plateau region into a “trough” in the υ vs [NADH] plot, whereas the “trough” of the υ vs [dichloroindophenol]plot was transformed into an intermediary plateau. Changes in the temperature of the assay system also led to changes in the kinetic curves. Incubation of the enzyme at 23 °C for 150 min caused disappearance of the intermediary plateau and “trough” regions in the υ vs [NADH]or [dichloroindophenol]plots, respectively. These appeared again after cooling at 0 ° C.The effect of pH, ionic strength, and temperature are discussed within the framework of two main models chosen by statistical analysis: (1) the reaction is catalyzed by a four-site enzyme exhibiting positive and negative cooperativity with respect to the catalytic (kinetic) activity of the sites involved; (2) two enzyme species each possessing two sites are present. These two are either independent or interconnected via a slow isomerization step. 相似文献
8.
Acetic acid formation in Escherichia coli fermentation 总被引:2,自引:0,他引:2
Theoretical analysis of cellulase product inhibition (by cellobiose and glucose) has been performed in terms of the mathematical model for enzymatic cellulose hydrolysis. The analysis showed that even in those cases when consideration of multienzyme cellulase system as one enzyme (cellulase) or two enzymes (cellulase and beta-glucosidase) is valid, double-reciprocal plots, usually used in a product inhibition study, may be nonlinear, and different inhibition patterns (noncompetitive, competitive, or mixed type) may be observed. Inhibition pattern depends on the cellulase binding constant, enzyme concentration, maximum adsorption of the enzyme (cellulose surface area accessible to the enzyme), the range in which substrate concentration is varied, and beta-glucosidase activity. A limitation of cellulase adsorption by cellulose surface area that may occur at high enzyme/substrate ratio is the main reason for nonlinearity of double-reciprocal plots. Also, the results of calculations showed that material balance by substrate, which is usually neglected by researchers studying cellulase product inhibition, must be taken into account in kinetic analysis even in those cases when the enzyme concentration is rather low. (c) 1992 John Wiley & Sons, Inc. 相似文献
9.
C Y Huang 《Archives of biochemistry and biophysics》1977,184(2):488-495
A new kinetic approach using alternative substrates as a tool for studying enzyme mechanisms is described. In this method the substrate to alternative substrate ratio is maintained constant and the common product (or summation of product analogs) is measured. The double-reciprocal plots so obtained at several constant ratios generate different patterns for various mechanisms, thus permitting a choice of kinetic model. In some cases, secondary intercept plots are utilized as a diagnostic aid. Another feature of this approach is that most of the resultant plots are linear. The graphical patterns for four cases of two-substrate, two-product reactions are presented as examples. These patterns allow one to differentiate several mechanisms which are not distinguishable by conventional alternative substrate, competitive inhibitor, or product inhibition studies alone. When used in combination with other methods, various mechanisms involving isomerization and abortive complex formation can be differentiated even if only one alternative substrate is available. 相似文献
10.
A convenient and accurate procedure for determining the kinetic parameter Vmax./Km is described. This avoids the error in the usual method of taking the observed first-order rate constant of an enzymic reaction at low substrate concentration as Vmax./Km. A series of reactions is used in which the initial concentration of substrate is below Km (e.g. from 5% to 50% of Km). Measurements are taken over the same extent of reaction (e.g. 70%) for each member of the series, and treated as if the kinetics were truly first-order. The reciprocal of the observed first-order rate constant is then plotted against the initial concentration of substrate: the reciprocal of the ordinate intercept is Vmax./Km. The procedure, as well as being applicable to simple reactions, is shown to be valid when there is competitive inhibition by the product, or when the reaction is reversible, or when there is competitive or mixed inhibition. The hydrolysis of cephalosporin C by a beta-lactamase from Pseudomonas aeruginosa is used to illustrate the method. 相似文献
11.
Takahiko Kato Kumiko Shimotohno 《Biochimica et Biophysica Acta (BBA)/General Subjects》1984,801(2):157-162
The method of kinetic analysis is developed to obtain the maximum velocity (Vm), the Michaelis constant (Km) and the parameters characterizing the inhibitors in an impure enzyme reaction, contaminated with one of four types of inhibitor (competitive, noncompetitive, uncompetitive and mixed-type). Although the reaction rate decreases with the increasing concentration of the enzyme sample containing an inhibitor, the double-reciprocal plot of the rate against the sample concentration becomes linear. The slopes of these linear plots at several different concentrations of substrate provide Km and the specific enzyme activity, which is proportional to Vm, in the sample. These linear straight lines intersect in a point, of which the coordinates give the unique parameters for the inhibitor. To prove the validity of this kinetic method, the model experiments were carried out with acetylcholinesterase and its inhibitors, phenyltrimethylammonium and trimethylammonium. The present method was applied to the measurement of the specific activity of galactosylceramide galactosidase in the mouse cerebral homogenate. In addition, a kinetic method is indicated for the inhibition of an enzymatic reaction by a contaminant which binds the substrate to reduce the fraction available to the enzyme. 相似文献
12.
The effect of viscosity on the rate of catalysis of carboxypeptidase A has been tested. By use of the tripeptide carbobenzoxy-l-alanyl-l-alanyl-l-alanine [Z(L-Ala)3] as substrate, it was shown that most of the effect on the hydrolysis rate caused by the presence of 30 or 40% methanol or glycerol in aqueous solution can be ascribed to a contribution of viscosity to the catalytic rate constant, kcat. Arrhenius plots of kcat in 30 and 40% glycerol or methanol are linear and almost parallel. When the rate constants are "corrected" for the viscosity of various media, the difference between the various Arrhenius plots is considerably reduced; it vanishes, within experimental error, when the effect of the dielectric constant of the solutions is taken into account as well. It is proposed that the viscosity of the medium can influence the rate-limiting step of the enzymic reaction, which is the rate of transitions over the energy barrier preceding product formation. According to the suggested mechanism, the enzyme--substrate complex can overcome this energy barrier by viscosity-dependent structural fluctuations. The quantitative agreement between the theory and the experimental results suggests that (a) due to the temperature dependence of the viscosity of the solution, the potential energy barrier of the reaction is about 5 kcal/mol lower than the observed activation energy and (b) information about the structural flexibility of the complex can be obtained by kinetic measurements. 相似文献
13.
14.
Jean M. Wardell William G. Bardsley John P. Kavanagh Reg M. Wood 《Journal of theoretical biology》1982,95(3):465-487
A number of details required for the classification of 3 : 3 double reciprocal plots are provided. It is shown that the ν(S) plot for a 3 : 3 function can have at most four inflexions and at most two inflexions adjacent to a turning point. Using this information, a classification of 3 : 3 ν(S) plots into ten main varieties with several subclasses is reported. The problem of defining the probability with which a given mechanism can give rise to specific curve shape features is considered. Applying this technique, the probability with which four simple enzyme mechanisms can give rise to 3 : 3 curve shapes is computed. It is shown that a 3 : 3 saturation function can have no turning points, at most two inflexions and at most one inflexion in double reciprocal space. The probability with which the available 3 : 3 shapes can arise is also computed. It is concluded that, with realistic values for rate constants, chemically reasonable enzyme mechanisms leading to rate equations of degree n : n can generate most of the kinetic profiles available to a rational function of degree n : n with positive coefficients. The probability of obtaining specific curve shapes is not so characteristic of the particular mechanism for 3:3 rate equations as it is for 2:2 rate equations. The probability of obtaining highly complex curves with several turning points or inflexions is rather lower for the enzyme mechanisms than with general 3 : 3 rational functions. There is a high probability that 3 : 3 mechanisms will generate kinetic curves that are geometrically similar to those possible for degree 2 : 2 but this is not so for binding isotherms. Hence differentiating 3 : 3 from 2 : 2 rate equations from experimental kinetic data is more likely to be successful by non-linear regression to the whole data set than by demonstrating a specific 3 : 3 feature. Binding curves, on the other hand, for three or more sites should give Scatchard plots with inflexions, features not possible with second degree equations which are conic sections in this space. 相似文献
15.
The initial development of a piosphere created by stocking a new watering trough with ca. 200 sheep at a near-pristine site in arid chenopod shrubland near Whyalla, South Australia, was studied. The following variables were monitored for up to 8 years using fenced plots as controls:shrub mortality; forage biomass and defoliation of the dominant shrubs and short-lived grasses; linear dimensions of Rhagodia ulicina; the density of short-lived species; and the invasion of exotics. Patterns in these variables were looked for which reflected the attenuation of stocking pressure with distance from water; that is, ‘piosphere patterns’. Piosphere patterns were detected for many variables. Those with values increasing towards the trough were mortality of the main forage shrub Atriplex vesicaria. the density of three short-lived taxa, viz. the bassias (Sclerolaena obliquicuspis, S. patenticuspis. Maireana sclerolaenoides), Tetragonia tetragonoides, and annual Zygophyllum spp. (Z. crenatum. Z. ovatum), and growth of short-lived grass species when sheep stocking pressure was low. Variables with values decreasing towards the trough were grass biomass, percentage of A. vesicaria individuals in flower, the percentage foliation of A. vesicaria and of the forage shrub Maireana sedifolia, and shrub forage biomass of A. vesicaria and M. sedifolia. The rate at which these patterns were registered varied. After only 3 months, a piosphere pattern was evident for grass biomass. Marrubium vulgare, an introduced weed, invaded the trough site within the first 6 months. Almost no A. vesicaria shrubs had died after 2 years, but after 8 years there was a marked piosphere pattern in their mortality. The piosphere patterns in forage biomass and defoliation of A. vesicaria and M. sedifolia became more pronounced with time. Some variables did not exhibit a piosphere pattern, such as the dimensions of the shrub R. ulicina and the densities of successive cohorts of the forb Erodium spp., even though there was an overall effect of grazing in reducing their values. There was no mortality of two dominant shrubs, M. sedifolia and R. ulicina, during the first 8 years. 相似文献
16.
《International biodeterioration & biodegradation》1999,43(1-2):57-61
The biodegradation kinetics for chlorinated aliphatic hydrocarbons trichloroethylene (TCE) by Burkholderia (Pseudomonas) cepacia PR131 and for1,2-dichloethane (1,2-DCA) by Xanthobacter autotrophicus GJ10 were determinedusing an initial rate method to determine the applicable rate law and relevant kinetic parametersunder aerobic conditions. A first order linear rate law applied to 1,2-DCA biodegradation by X. autotrophicus GJ10. The first order rate constant was determined to be 0.014 ml/min/mg.A non-linear rate law applied to TCE biodegradation by B. cepacia PR131.The maximum specific degradation rate constant was determined to be 0.8 nmol/min/mg protein,and the half saturation constant was determined to be 0.026 mM (3.47 ppm). Error analysisperformed on our analytical methods and computations, using a logarithmic differentiationmethod, indicated the relative error of our reported rate constants to be approximately 17%.Knowledge of the kinetic rate laws and kinetic parameters governing the biodegradation of TCEand 1,2-DCA by these strains will further the application of these strains in the environmental fieldand in waste treatment applications. 相似文献
17.
The effect of mass transfer resistances on the Lineweaver-Burk plots in immobilized enzyme systems has been investigated numerically and with analytical approximate solutions. While Hamilton, Gardner & Colton (1974) studied the effect of internal diffusion resistances in planar geometry, our study was extended to the combined effect of internal and external diffusion in cylindrical and spherical geometries as well. The variation of Lineweaver-Burk plots with respect to the geometries was minimized by modifying the Thiele modulus and the Biot number with the shape factor. Especially for a small Biot number all the three Lineweaver-Burk plots fell on a single line. As was discussed by Hamilton, et al (1974), the curvature of the line for large external diffusion resistances was small enough to be assumed linear, which was confirmed from the two approximate solutions for large and small substrate concentrations. Two methods for obtaining intrinsic kinetic constants were proposed: First, we obtained both maximum reaction rate and Michaelis constant by fitting experimental data to a straight line where external diffusion resistance was relatively large, and second, we obtained Michaelis constant from apparent Michaelis constant from the figure in case we knew maximum reaction rate a priori. 相似文献
18.
Robert P. Jaques 《Journal of invertebrate pathology》1974,23(2):140-152
Concentrations of the nuclear-polyhedrous virus (T. ni NPV) and the granulosis virus (T. ni GV) of the cabbage looper, Trichoplusia ni, in soil and on foliage were monitored up to 4 years after treatment.A single application of T. ni NPV to soil in August or 5 foliar applications of the virus at 10-day intervals in August and early September maintained substantial concentrations of the virus on foliage and high concentrations of the virus accumulated in soil. With development of natural epizootics of the virus disease in populations of the host larvae in September and October, substantial concentrations of the virus accumulated in soil and on foliage in nontreated plots, eventually becoming equal in amount with the virus in virus-treated plots. The virus accumulated more slowly in plots treated with chemical insecticides or Bacillus thuringiensis because few host larvae survived to support late-season epizootics of the disease. Small quantities of T. ni NPV were detected in heads of cabbage harvested from the plots in October.Long-term studies in which nontreated plots and plots treated with T. ni NPV or T. ni GV were replanted for up to 4 years after treatment showed that concentrations of T. ni NPV in surface soil remained constant during the winter but were reduced by dilution during cultivation preparatory to planting in the spring. T. ni NPV accumulated during the late summer and autumn with development of epizootics of the disease in populations of host larvae. Increased concentrations of the virus in soil coincided with increased concentrations on leaves in each year. T. ni GV did not persist on leaves or in soil following application and only small amounts were found 2 years after application.T. ni NPV disease was prevalent in September and October in populations of host larvae in plots in which substantial residues of the virus were found. These epizootics contributed substantially to late-season control of the looper after completion of spraying. 相似文献
19.
The kinetic rate equation for the photosensitized cleavage reaction of surface-bound photolabile chromophores with free diffusion of sensitizer molecules from the bulk of a solution to the surface is derived by determining the stationary solution of a diffusion equation with suitable boundary conditions. The relation between the phenomenological rate constant for the photosensitized reaction at the surface and in the bulk is established. Applying the result to the analysis of an experimental example, the origin of the quasi zeroth-order kinetics of the sensitized reaction is revealed. A theoretical comparison of intramolecular sensitization in photocleavable protecting groups with a molecular antenna and sensitization with the freely diffusing sensitizer shows that in a typical case sensitization with free diffusion is more effective than intramolecular sensitization for sensitizer concentrations higher than 5 mM. 相似文献
20.
Vicarious nucleophilic substitution reactions are a versatile way of introducing substituents into aromatic and heteroaromatic electron-deficient compounds. In this project, a kinetic study of these reactions by applying quantum mechanics concepts, such as reaction force, force constant, and electronic reaction flow was proposed. Furthermore, absolute theoretical scales of electrophilicity by applying density functional theory electronic indices were established to classify a series of five and six-membered nitroheteroarenes, and nitrobenzenes with substituents in ortho, meta and para positions. The theoretical model was validated by comparison with experimental kinetic results. Calculations using B3LYP/6-311G(d,p) level of theory allowed analysis of the reactivity patterns and the mechanisms of these chemical reactions. The theoretical scale properly accounts for the activating/deactivating effects promoted by the substituents and agrees with the ability of these substituents to accept or donate electrons, electron acceptor substituents are those that increase electrophilicity, and electron donors those that reduce it. 相似文献