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1.
Information about ligand binding, dissociation, internalization, and intracellular processing and about receptor turnover, processing, and insertion into the membrane is contained in the time-dependent changes in concentrations of membrane-associated and internalized ligand. Single experiments similar in design to those typically performed for Scatchard analyses of binding data conducted at physiological temperature and in the absence of inhibitors of ligand-receptor complex internalization and degradation can provide kinetic data sufficient to permit derivation of all the respective rate constants by numerical methods. We developed an analytical solution of the kinetic model which assumes that all of these processes follow first order kinetics. The model represents interactions of surface receptors (R)s, the surface ligand-receptor complex (LR)s and internalized receptor-ligand complex (LR)I: d[R]S/dt = Vr - kt[R]S - ka[L] [R]S + kd [LR]S; d[LR]S/dt = ka[L] [R]S - kd[LR]S - ke[LR]S; d[LR]I/dt = ke[LR]S - kh[LR]I; Vr is the constant rate of insertion of receptors into the membrane, kt is the internalization rate constant for free receptors, ka and kd are association and dissociation rate constants for ligand-surface receptor interaction, ke is the internalization rate constant for ligand-receptor complexes, and kh is the intracellular ligand decomposition rate constant. The interaction of radioiodinated human recombinant interferon-alpha 2a with the human alveolar lung carcinoma cell line, A549, was adequately accounted for by the model. The rate constants, numerically derived from time-dependent concentrations of surface-bound and internalized ligand of other systems taken from the literature, were in agreement with values of these rate constants individually measured by steady-state experiments. In cases where the fate of internalized radioactivity was more complex than assumed by the model, the parameters ka, kt, (kd + ke) and Vr could be derived from the time dependence of [LR]S.  相似文献   

2.
We measured the kinetic parameters for interaction of epidermal growth factor (EGF) with fetal rat lung (FRL) cells under two sets of experimental conditions and applied sensitivity analysis to see which parameters were well-defined. In the first set of experiments (method 1), the kinetics of internalization and dissociation of radiolabeled EGF were measured with a temperature-shift protocol in medium initially devoid of free ligand. The initial concentration of radiolabeled EGF bound to the cell surface corresponded to levels of receptor occupancy ranging from approximately 200 receptors per cell to approximately 18,000 receptors per cell, a level at which EGF binding approaches saturation. In the second set of experiments (method 2), carried out at a constant temperature, we began with no surface-bound or internalized ligand. The initial free ligand concentration was varied from 0.2 to 50 ng/mL. In both sets of experiments, we measured surface-bound, internalized, and free 125I-EGF as functions of time and evaluated the parameters of a mathematical model of endocytosis. Sensitivity analysis showed that three rate constants were well-defined in this combination of two experimental approaches: ke, the endocytic rate constant; ka, the association rate constant; and kd, the dissociation rate constant. The endocytic parameter ke was found to be independent of initial surface receptor occupancy (method 1); there was some indication that it increased with initial free ligand concentration in method 2. Neither kd nor ka was found to change with extent of initial surface receptor occupancy or initial free ligand concentration, respectively, a finding of significance, since diffusion theory predicts these parameters will vary with surface receptor occupancy.(ABSTRACT TRUNCATED AT 250 WORDS)  相似文献   

3.
Pharmacokinetics of pralidoxime chloride in the rat   总被引:2,自引:0,他引:2  
M D Green  B G Talbot  C R Clark 《Life sciences》1986,39(23):2263-2269
The pharmacokinetics of pralidoxime chloride (2-PAM) was studied in rats. Different groups of rats were given an intramuscular injection of 2-PAM at one of three doses (20, 40, or 80 mg/kg). This range of doses is used commonly in studies concerned with the efficacy of 2-PAM against poisoning by potent organophosphorus inhibitors of cholinesterase enzyme. Individual, sequential blood samples were collected during the course of the experiment. From these blood samples the plasma concentrations of 2-PAM were determined over time for each animal. Next the relationship of plasma concentration to time was expressed in terms of a standard pharmacokinetic model. Estimates of various pharmacokinetic parameters were calculated using an open, one-compartment model: volume of distribution (Vd), maximal plasma concentration (Cmax), elimination rate constant (k10), absorption rate constant (k01), area under the curve (AUC) and clearance (CL). Of the pharmacokinetic estimates, only Cmax and AUC were found to be statistically significant (p less than 0.0001) when compared across all the doses; these pharmacokinetic estimates were highly correlated with doses with r = 0.998 and r = 0.997, respectively. However, when AUC and Cmax were normalized by dividing through by dose, no significant differences were found in the transformed data. The results of this study in rat indicate that the pharmacokinetics of 2-PAM is linearly related to dose in a range employed in therapeutic studies of 2-PAM.  相似文献   

4.
The E5 protein of the bovine papillomavirus induces cellular transformation when transfected into NIH 3T3 cells, and the extent of focal transformation is enhanced by cotransfection with the epidermal growth factor (EGF) receptor (Martin et al., Cell 59:21-32, 1989). To determine whether E5 affects EGF:receptor interactions we analyzed the kinetics of 125I-EGF processing using a mathematical model that enabled us to evaluate rate constants for ligand association (ka), dissociation (kd), internalization (ke), recycling (kr), and degradation (kh). These rate constants were measured in NIH 3T3 cells transfected with the human EGF receptor (ER cells) and in cells transfected with both the EGF receptor and E5 (E5/ER cells). We found that the rate constant for 125I-EGF association ka was significantly decreased in E5/ER cells, but was apparently occupancy-independent in both cell lines. The 125I-EGF dissociation rate constant kd was significantly lower in E5 transformed cells, and increased with occupancy in both cell lines. This suggests that E5 alters the receptor before or during EGF binding so that ligand association is slower; however, once complexes are formed, EGF is bound more tightly to the receptor. Rate constants for internalization ke were also found to be occupancy-dependent, although at a given level of occupancy ke was similar for both cell lines. Also, there was no apparent effect of E5 on the recycling rate constant kr. The 125I-EGF degradation rate constant kh was 30% lower in E5 transformed cells, and was occupancy-independent. The overall effect of E5 is to stabilize intact EGF:receptor complexes which may alter mitogenic signaling of the receptor.  相似文献   

5.
Resolution of kinetic equations and parameter identification are discussed for n-compartment linear catenary models with elimination allowed from any compartment. For a given input, general formulas are derived to describe the tracer amount in any compartment as a function of the model parameters. Conversely, explicit procedures are given to identify the model parameters when the concentration-time curve is known in one arbitrary compartment, the tracer being injected into the same compartment. In this inverse problem, the solution is not unique: the model transfer rate constants can only be localized in a finite set of intervals.  相似文献   

6.
Decreased binding capacity of the erythrocyte complement receptor (RBC CR1) in systemic lupus erythematosus (SLE) may contribute to abnormal handling of circulating immune complexes in these patients. Decreased numbers of RBC CR1 have been reported in SLE, but, since binding is a function of both receptor number and receptor binding kinetics, we measured kinetic parameters for the interaction of complement (C) containing [3H]DNA:anti-DNA immune complexes (IC) with normal control (NC) and SLE RBC. Experiments were performed at five temperatures ranging from 7-37 degrees C. The parameters measured included: (1) the maximum quantity of DNA:anti-DNA:C which could bind per RBC, S; (2) the association rate constant, ka, for the binding of DNA:anti-DNA:C to RBC; (3) the dissociation rate constant, kd, for the dissociation of bound DNA:anti-DNA:C IC from RBC; (4) the steady-state constant, Kss (ka/kd); and (5) the energies of activation for association, Eaa, and dissociation, Ead. Although the relative amount of bound DNA:anti-DNA:C per RBC was significantly decreased in SLE patients compared to NC (P less than 0.001), the mean values for Kss, ka, kd, Eaa and Ead did not differ significantly between the two groups. These data suggest the following: (1) RBC CR1 binding and dissociation of DNA:anti-DNA:C are consecutive reactions resulting in steady-state concentrations of free and RBC-bound IC; (2) at steady-state times, the ratio of RBC bound to unbound DNA:anti-DNA:C are governed by kinetic factors; (3) since the binding kinetics of SLE and NC RBC are not significantly different, the decreased binding activity described by other investigators can only be due to a decreased number of CR1 per RBC; and (4) values for Eaa and Ead suggest that the rate-determining steps in IC association with and dissociation from RBC involves making and breaking of hydrogen bonds.  相似文献   

7.
The release by glycyl-L-phenylalanine 2-naphthylamide (Gly-L-Phe-2-NNap) of endocytosed invertase associated with the MLP fraction (sum of the M, L and P fractions [de Duve, Pressman, Gianetto, Wattiaux & Appelmans (1955) Biochem. J. 63, 604-617]) of rat liver was investigated and compared with the release of cathepsin C. The percentage of invertase released increases with time after the enzyme injection, whereas the release of cathepsin C is not influenced by this treatment and corresponds to 85-90% of the total activity of the enzyme. It takes about 2h to attain a similar release of both enzymes. The quantity of invertase releasable or not by Gly-L-Phe-2-NNap was plotted against the time after the injection. Results agree well with the hypothesis that unreleasable invertase is associated with a pre-lysosomal compartment, whereas releasable invertase is present in lysosomes. A kinetic analysis indicates that invertase enters the pre-lysosomal compartment with a zero-order rate constant of 0.48 unit/min per g fresh wt., and leaves this compartment with a first-order rate constant of 0.042 min-1.  相似文献   

8.
The classic solutions based on specific activity curves for the kinetic analysis of 45Ca movements in three compartment cellular systems cannot be used when the extracellular compartment is one to two orders of magnitude larger than the cellular or tissue compartments. However if the relative radioactivity curve (tracer uptake curve) is analyzed it is possible to calculate all the relevant kinetic parameters. This paper offers the solutions based on relative radioactivity measurements for the calculation of exchange rates, rate constants and compartment sizes of three compartment systems, for series and parallel cases, for closed and open systems.  相似文献   

9.
Kinetic analysis of the time course of plasma TSH after TRH stimulation was performed by means of a single compartment model with first order input. This kinetic model showed satisfactory fit to the data, and was found to be useful enough for the characterization of plasma TSH dynamics. In endocrinologically normal short children, the amount of TSH release per unit volume of distribution space (Q0/V), the rate constant for the TSH release (alpha), and the rate constant for TSH elimination (beta) were averaged 23.4 microU/ml, 6.981 hr-1 and 0.813 hr-1, respectively. Elevated Q0/V values with lowered alpha and beta were obtained in the hypothyroid children. Variable results, with the exception of low alpha values, were obtained in the children with pituitary dwarfism.  相似文献   

10.
W P Lu  Y Sun  M D Bauer  S Paule  P M Koenigs  W G Kraft 《Biochemistry》1999,38(20):6537-6546
Penicillin-binding protein 2a (PBP2a) is the primary beta-lactam resistance determinant of methicillin-resistant Staphylococcus aureus (MRSA). MecA, the gene coding for PBP2a, was cloned with the membrane-anchoring region at the N-terminus deleted. The truncated protein (PBP2a) was overexpressed in Escherichia coli mostly in the soluble form accounting for approximately 25% of soluble cell protein and was purified to homogeneity. The purified protein was shown to covalently bind beta-lactams in an 1:1 ratio as determined by electrospray mass spectrometry. A novel method based on HPLC-elctrospray mass spectrometry has been developed to quantitatively determine the formation of the covalent adducts or acyl-PBP2a complexes. By using this method, combined with kinetic techniques including quench flow, we have extensively characterized the interactions between PBP2a and three beta-lactams and determined related kinetic parameters for the first time. The apparent first-order rate constants (ka) of PBP2a acylation by benzylpenicillin showed a hyperbolic dependence on the concentration of benzylpenicillin. This is consistent with the mechanism that the binding of the penicillin to PBP2a consists of reversible formation of a Michaelis complex followed by formation of the penicilloyl-PBP2a adduct, and allowed the determination of the individual kinetic parameters for these two steps, the dissociation constant Kd of 13.3 mM and the first-order rate constant k2 of 0.22 s-1. From these values, the second-order rate constant k2/Kd, the value reflecting the overall binding efficiency of a beta-lactam, of 16.5 M-1 s-1 was obtained. The fairly high Kd value indicates that benzylpenicillin fits rather poorly into the protein active site. Similar studies on the interaction between PBP2a and methicillin revealed k2 of 0.0083 s-1 and Kd of 16.9 mM, resulting in an even smaller k2/Kd value of 0.49 M-1 s-1. The rate constants k3 for deacylation of the acyl-PBP2a complexes, the third step in the interactions, were measured to be <1.5 x 10(-)5 s-1. These results indicate that the resistance of PBP2a to penicillin inactivation is mainly due to the extremely low penicillin acylating rate in addition to the low association affinity, but not to a fast rate of deacylation. Acylation of PBP2a by a high-affinity cephalosporin, Compound 1, also followed a saturation curve of ka versus the compound concentration, from which k2 = 0.39 s-1, Kd = 0.22 mM, and k2/Kd = 1750 M-1 s-1 were obtained. The 100-fold increase in the k2/Kd value as compared with that of benzylpenicillin is mostly attributable to the decreased (60-fold) Kd, indicating that the cephalosporin fits much better to the binding pocket of the protein.  相似文献   

11.
Dopamine Uptake by Rat Striatal Synaptosomes: A Compartmental Analysis   总被引:5,自引:3,他引:2  
Abstract: Dopamine (DA) uptake into synaptosomes from rat corpus striatum was studied in the presence of a monoamine oxidase (MAO) inhibitor and dithiothreitol, by means of a filtration technique. Under these conditions a steady state develops rapidly in which the synaptosomal DA content remains constant while the continuing DA uptake is counterbalanced by DA efflux from the synaptosome. Exchange of synaptosomal [3H]DA and [14C]DA was measured under these conditions. In timecourse experiments it was found that exchange could be described significantly better by a three-compartment model than by a two-compartment model. However, if synaptosomes from reserpine-pretreated animals were used, analysis according to a three-compartment model did not result in a significantly better fit compared with a two-compartment model. Subsequently, kinetic transfer parameters describing DA fluxes between compartments at different DA concentrations were calculated from the fitted exchange curves. A Michaelis-Menten kinetic analysis indicated that only the in-series three-compartment configuration, in which DA is taken up from the medium into one synaptosomal compartment, from which it can subsequently be transferred to a second compartment without direct access to the medium, gave kinetically acceptable results. Transfer parameters in synaptosomes from reserpine-treated rats were comparable to those parameters describing DA transport between the medium and the first intrasynaptosomal compartment as measured under control conditions. Morover, it was found that potassium depolarization of synaptosomes resulted in a release of DA in a quantity similar to that found in the second intrasynaptosomal compartment. It is suggested that the two intrasynaptosomal compartments found correspond to a cytoplasmatic and vesicular DA pool, respectively. The functional significance of these findings is discussed in terms of the regulation of DA levels within the nerve terminal.  相似文献   

12.
《MABS-AUSTIN》2013,5(1):49-55
AUC, area under the concentration-time curve; CGN, chronic glomerulonephritis; Cl, Clearance rates; Cmax, maximum serum concentration; CsA, cyclosporin A; CV, coefficient of variation; DGF, delayed graft function; ELISA, enzyme-Linked immunosorbent assay; ESRD, end-stage renal disease; FITC, fluorescein isothiocyanate; HRP, horseradish peroxidase; LOQ, limit of quatitation; MMF, mycophenolate mofetil; PD, pharmadynamics; PE, phycoerythrin; PK, pharmacokinetics; QC, quality control; SAE, serious adverse event; T1/2ke, terminal half-life; TBS, bovine serum albumin in tris buffered saline; Tmax, time to reach the peak concentration; TBST, Tris-Buffered Saline Tween-20; Vd, volumes of distribution  相似文献   

13.
14.
Even though the glycine conjugation pathway was one of the first metabolic pathways to be discovered, this pathway remains very poorly characterized. The bi‐substrate kinetic parameters of a recombinant human glycine N‐acyltransferase (GLYAT, E.C. 2.3.1.13) were determined using the traditional colorimetric method and a newly developed HPLC–ESI‐MS/MS method. Previous studies analyzing the kinetic parameters of GLYAT, indicated a random Bi–Bi and/or ping‐pong mechanism. In this study, the hippuric acid concentrations produced by the GLYAT enzyme reaction were analyzed using the allosteric sigmoidal enzyme kinetic module. Analyses of the initial rate (v) against substrate concentration plots, produced a sigmoidal curve (substrate activation) when the benzoyl‐CoA concentrations was kept constant, whereas the plot with glycine concentrations kept constant, passed through a maximum (substrate inhibition). Thus, human GLYAT exhibits mechanistic kinetic cooperativity as described by the Ferdinand enzyme mechanism rather than the previously assumed Michaelis–Menten reaction mechanism.  相似文献   

15.
Previously, we have shown, in vivo, that the acyl coenzyme A: cholesterol acyltransferase (ACAT) inhibitor avasimibe decreases hepatic apolipoprotein (apo) B secretion into plasma. To test the hypothesis that avasimibe modulates postprandial triglyceride-rich lipoprotein (TRL) metabolism in vivo, an oral fat load (2 g fat/kg) containing retinol was given to 9 control miniature pigs and to 9 animals after 28 days treatment with avasimibe (10 mg/kg/day, n=5; 25 mg/kg/day, n=4). The kinetic parameters for plasma retinyl palmitate (RP) metabolism were determined by multi-compartmental modeling using SAAM II. Avasimibe decreased the 2-h TRL (d<1.006 g/mL; S(f)>20) triglyceride concentrations by 34%. The TRL triglyceride 0-12 h area under the curve (AUC) was decreased by 21%. In contrast, avasimibe had no effect on peak TRL RP concentrations, time to peak, or its rate of appearance into plasma, however, the TRL RP 0-12 h AUC was decreased by 17%. Analysis of the RP kinetic parameters revealed that the TRL fractional clearance rate (FCR) was increased 1.4-fold with avasimibe. The TRL RP FCR was negatively correlated with very low density lipoprotein (VLDL) apoB production rate measured in the fasting state (r=-0.504). No significant changes in total intestinal lipid concentrations were observed. Thus, although avasimibe had no effect on intestinal TRL secretion, plasma TRL clearance was significantly increased; an effect that may relate to a decreased competition with hepatic VLDL for removal processes.  相似文献   

16.
1. The results of kinetic studies in vitro are difficult to apply to metabolic reactions in vivo. 2. In living vertebrates reaction rates are usually first-order and for a particular reaction the rate "constant", k, varies with the several thousand-fold variations in metabolic rate. 3. Therefore, in the kinetic equation (formula: see text) since K varies with metabolic rate, Vmax and/or Km will aslo vary. 4. However, reaction rates for a series of different substrates were similar in animals varying widely in metabolic rate if corrections were made for differences in blood flow. 5. The observation that metabolic rate (reaction rate) is directly dependent on blood flow (Coulson et al., 1977) allowed derivation of new kinetic constants which were valid in vivo. 6. Introduction of a flow term into the observed first-order equations yields an "affinity constant", K, or a "flow constant", KF, somewhat analogous to Km, which allows one to predict reaction rates in animals of different metabolic rates. 7. The defining equations are (formula: see text) where V = reaction velocity in mmol/hr/kg tissue, [S] = millimolar substrate concentration in the blood, k = In 2/tau (tau the half-life) and F = blood flow in 1/hr/kg tissue. 8. Thus, K = k/F = 1/KF. The KF's for the initial step in the degradation of 17 amino acids in rats, dogs, lizards, turtles and alligators were similar, demonstrating the similarity of enzyme affinities in different species.  相似文献   

17.
We have analyzed by (31)P MRS the relationship between kinetic parameters of phosphocreatine (PCr) recovery and end-of-exercise status under conditions of moderate and large acidosis induced by dynamic exercise. Thirteen healthy subjects performed muscular contractions at 0.47 Hz (low frequency, moderate exercise) and 0.85 Hz (high frequency, heavy exercise). The rate constant of PCr resynthesis (k(PCr)) varied greatly among subjects (variation coefficients: 43 vs. 57% for LF vs. HF exercises) and protocols (k(PCr) values: 1.3+/-0.5 min(-1) vs. 0.9+/-0.5 min(-1) for LF vs. HF exercises, P<0.03). The large intersubject variability can be captured into a linear relationship between k(PCr), the amount of PCr consumed ([PCr(2)]) and pH reached at the end of exercise (pH(end)) (k(PCr)=-3.3+0.7 pH(end)-0.03 [PCr(2)]; P=0.0007; r=0.61). This dual relationship illustrates that mitochondrial activity is affected by end-of-exercise metabolic status and allows reliable comparisons between control, diseased and trained muscles. In contrast to k(PCr), the initial rate of PCr recovery and the maximum oxidative capacity were always constant whatever the metabolic conditions of end-of-exercise and can then be additionally used in the identification of dysfunctions in the oxidative metabolic pathway.  相似文献   

18.
A kinetic study has been made of the release ofd-aspartate from rat cortical synaptosomes following pre-loading with labelled D-aspartate, and the results compared to a previous study of the release of the acidic amino acids glutamate plus aspartate following pre-loading with labeledl-glutamate. Qualitatively, the results of the two studies are similar. The D-aspartate taken up during the preload period appears to be totally releasable. However, release is greatly increased by depolarizing media. The increased rate of release induced by increasing [K]o is independent of the [Ca]o, while veratrine-induced release is inhibited by [Ca]o. Release is from more than a single compartment, since plots of the log10 of the synaptosomal D-aspartate content (calculated from the label content) as a function of the incubation time are non-linear for all incubation solutions. In the previous study which utilizedl-glutamate pre-loading, the results were consistent with either a model consisting of two passive compartments (that is, synaptosomal content T as a function of time is given by Ae–Kat+Be–Kbt, in which A and B are compartment sizes, Ka and Kb are exchange constants, and t is incubation time) or a model consisting of one passive compartment (Ae–Kat) and one saturated carrier compartment (T-Kbt, in which T=total content at zero time and Kb=maximal velocity). The present results withd-aspartate also give excellent fits to these models. However, there are some quantitative differences in the estimates of the compartment sizes and exchange constants, which are obtained by optimizing the fit of the data to the equation for each model. Although most of these quantitative differences appear to be minor, one difference between the two studies is of potential significance in interpretation of the results. In the glutamate study, all depolarizing media were found to reduce the exchange constant for the carrier mechanism, while in the present study, depolarizing media were found to increase the exchange constant, with the exception of veratrine-containing medium without calcium.  相似文献   

19.
Among the various cardiac contractility parameters, left ventricular (LV) ejection fraction (EF) and maximum dP/dt (dP/dt(max)) are the simplest and most used. However, these parameters are often reported together, and it is not clear if they are complementary or redundant. We sought to compare the discriminative value of EF and dP/dt(max) in assessing systolic dysfunction after myocardial infarction (MI) in swine. A total of 220 measurements were obtained. All measurements included LV volumes and EF analysis by left ventriculography, invasive ventricular pressure tracings, and echocardiography. Baseline measurements were performed in 132 pigs, and 88 measurements were obtained at different time points after MI creation. Receiver operator characteristic (ROC) curves to distinguish the presence or absence of an MI revealed a good predictive value for EF [area under the curve (AUC): 0.998] but not by dP/dt(max) (AUC: 0.69, P < 0.001 vs. EF). Dividing dP/dt(max) by LV end-diastolic pressure and heart rate (HR) significantly increased the AUC to 0.87 (P < 0.001 vs. dP/dt(max) and P < 0.001 vs. EF). In na?ve pigs, the coefficient of variation of dP/dt(max) was twice than that of EF (22.5% vs. 9.5%, respectively). Furthermore, in n = 19 pigs, dP/dt(max) increased after MI. However, echocardiographic strain analysis of 23 pigs with EF ranging only from 36% to 40% after MI revealed significant correlations between dP/dt(max) and strain parameters in the noninfarcted area (circumferential strain: r = 0.42, P = 0.05; radial strain: r = 0.71, P < 0.001). In conclusion, EF is a more accurate measure of systolic dysfunction than dP/dt(max) in a swine model of MI. Despite the variability of dP/dt(max) both in na?ve pigs and after MI, it may sensitively reflect the small changes of myocardial contractility.  相似文献   

20.
The displacement of NADH from cytoplasmic aldehyde dehydrogenase (EC 1.2.1.3) from sheep liver was studied by using NAD+, 1,10-phenanthroline, ADP-ribose, deamino-NAD+ and pyridine-3-aldehyde-adenine dinucleotide as displacing agents, by following the decrease in fluorescence as a function of time. The data obtained could be fitted by assuming two first-order processes were occurring, a faster process with an apparent rate constant of 0.85 +/- 0.20 s-1 and a relative amplitude of 60 +/- 10% and a slower process with an apparent rate constant of 0.20 +/- 0.05 s-1 and a relative amplitude of 40 +/- 10% (except for pyridine-3-aldehyde-adenine dinucleotide, where the apparent rate constant for the slow process was 0.05 s-1). The displacement rates did not change significantly when the pH was varied from 6.0 to 9.0. Kinetic data are also reported for the dependence of the rate of binding of NADH to the enzyme on the total concentration of NADH. Detailed arguments are presented based on the isolation and purification procedures, the equilibrium coenzyme-binding studies and the kinetic data, which lead to the following model for the release of NADH from the enzyme: (formula: see article). The parameters that best fit the data are: k + 1 = 0.2 s-1; k - 1 = 0.05 s-1; k + 2 = 0.8 s-1 and k - 2 = 5 X 10(5)litre-mol-1-s-1. The slow phase of the NADH release is similar to the steady-state turnover number for substrates such as acetaldehyde and propionaldehyde and appears to contribute significantly to the limitation of the steady-state rate.  相似文献   

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