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At least 667 records · Page 37Linked to original sources

PubMed2002

Comparison of oxygen uptake on-kinetic calculations in heart failure.

PURPOSE: The analysis of oxygen (O(2)) uptake on-kinetics during steady-rate is gaining interest in the heart failure (HF) population. The rate change in O(2) at the initiation of exercise can be assessed via nonlinear regression time constant (TC) or an algebraic equation (mean response time [MRT]). These calculations are presumed to be interchangeable, but research supporting this claim is limited. This investigation compares and contrasts two of the more commonly used O(2) uptake on-kinetic calculations. METHOD: Twenty-eight subjects diagnosed with compensated HF and 19 age, sex, and activity-matched controls underwent a symptom-limited exercise test and a steady-rate exercise session (6 min). Peak O(2) uptake, O(2) uptake at ventilatory threshold, the O(2) uptake TC (TC), and the O(2) uptake mean response time (MRT) were calculated for each subject. RESULTS: O(2) uptake on-kinetics was significantly faster for the control group ( < 0.05) regardless of calculation method. There was a significant difference between the O(2) uptake TC and MRT for the HF group. All O(2) uptake on-kinetic calculations were significantly correlated with aerobic capacity. CONCLUSIONS: O(2) uptake TC and MRT values may not be interchangeable in the HF population. All O(2) uptake on-kinetic calculations did produce a significant difference between experimental and control groups and correlated with indicators of aerobic capacity. The 10-s O(2) uptake on-kinetic calculations may be preferable secondary to expired gas fluctuations associated with breath-by-breath measures. Further work is, however, needed to determine which averaged O(2) uptake on-kinetic expression is optimal given the significant difference between TC and MRT. A mechanism for this difference may be the oscillatory ventilatory expired gas pattern demonstrated by some patients with HF.

Adult↗

PubMed1976

Dissociation kinetics of complexes between the antibiotic rifamycin and DNA-dependent RNA polymerase from Escherichia coli.

Phosphocellulose chromatography was employed to measure the binding of 3-(2-[14C]acetamidoethyl)-thiorifamycin(abbreviated[14C]AcNHEtS-Rif) to RNA polymerase core enzyme. The technique yielded a correct value for the stoichiometry of interaction. The same method was successfully applied to an investigation of the dissociation kinetics of the [14C]AcNHEtS-Rif - core-polymerase complex. We observed biphasic dissociation kinetics not in agreement with the existence of one single first-order dissociation step. Assuming two independent dissociation reactions, rate constants have been evaluated differing roughly by 10-fold (1.7 X 10(-3) s-1 and 1.5 X 10(-4) s-1 at 25 degrees C). The ratio of the amplitudes of the biphasic dissociation kinetics changed with temperature. The kinetic data are interpreted as indirect evidence for the existence of two enzyme species differing in their dynamic properties with respect to the binding of the antibiotic rifamycin. Our data furthermore lend support to the assumption that the two enzyme forms are in equilibrium. The observed sigmoidal dependence of the dissociation rate on temperature could indicate a conformational transition of RNA core polymerase with a transition midpoint at around 20 degrees C. The dye, rose bengal, was found to be as effective as AcNHEtS-Rif itself as a competing agent. The dissociation kinetics of the [14C]-AcNHEtS-Rif - core-polymerase complex in the presence of an excess of the dye rose bengal were found to be very similar to those measured in the presence of AcNHEtS-Rif. This could mean that rose bengal and AcNHEtS-Rif compete for the same site at RNA core polymerase. The dissociation of the ternary complex [14C]AcNHEtS-Rif - core-polymerase - poly[d(A-T)] was followed by gel filtration. Up to the extent of dissociation measured, the reaction appeared to follow first-order kinetics. The dissociation rate constant was calculated to be 1.7 X 10(-4) s-1. Experiments to determine the effect of the antibiotic streptolydigin on the dissociation kinetics of the [14C]AcNHEtS-Rif - core-polymerase complex have not led to conclusive results.

Binding Sites↗

PubMed1986

Kinetic implications of the occurrence of several relaxations in the conformational transition of mnemonical enzymes.

If the conformational transition involved in enzyme memory occurs in several elementary steps, the time constant of the overall 'slow' relaxation is mostly determined by the individual values of the rate constants pertaining to the overall transconformation. The extent of kinetic co-operativity of the enzyme reaction, however, is mostly controlled by the degree of reversibility of the elementary steps of the conformational transition. There is then no simple relation between the time scale of the 'slow' transition and the extent of kinetic co-operativity of the enzyme reaction. A slow transition of about 10(-3) s-1 is therefore perfectly compatible with a strong positive or negative co-operativity and in particular with the negative co-operativity observed with wheat germ hexokinase LI. The relationship that has been established recently [Pettersson, G. (1986) Eur. J. Biochem. 154, 167-170] between the 'slow' enzyme relaxation and the extent of kinetic co-operativity holds only in the specific case where the transconformation occurs in one step. Owing to the possible occurrence of a multistep conformation change, the lack of this relationship means nothing as to the validity, or the invalidity, of the concept of mnemonical transition. More informative than the time scale of the 'slow' transition is its dependence with respect to glucose and glucose 6-phosphate, which both react with the enzyme. The effect of reaction products on the modulation of kinetic co-operativity is also of cardinal importance in the diagnosis of enzyme memory. Since an alternative model has been recently proposed by Pettersson (cited above) to explain the mechanistic origin of kinetic co-operativity of monomeric enzymes, the effect of products on the kinetic co-operativity predicted by this alternative model has been studied theoretically, in order to determine whether it is consistent with the experimental results obtained with wheat germ hexokinase LI. This analysis shows that the predictions of this model are in total disagreement with both the predictions of the mnemonical model and the experimental results obtained with wheat germ hexokinase LI, as well as with other enzymes. This alternative model cannot therefore be considered as a sensible explanation of the mechanistic origin of co-operativity of monomeric enzymes. It is therefore concluded that the mnemonical model which rests on numerous experimental results, obtained by different research groups, on different enzymes is the simplest and most likely explanation of the kinetic subtleties displayed by some monomeric enzymes, and in particular wheat germ hexokinase LI.

Enzymes↗

PubMed1990

Thermodynamics of information transfer between subunits in oligomeric enzymes and kinetic cooperativity. 3. Information transfer between the subunits of chloroplast fructose bisphosphatase.

The theory and the methods that have been described in the two preceding papers in this journal have been used to analyze the kinetic properties of chloroplast fructose bisphosphatase. The enzyme is a tetramer made up of apparently identical subunits and displays a sigmoidal kinetics with respect to its substrate, fructose bisphosphate. The free ionic species, magnesium and fructose bisphosphate bind to the enzyme and the chelate fructose-bisphosphate-magnesium does not affect the sigmoidicity of the rate curves. The Hill coefficient with respect to free fructose bisphosphate is equal to 2.3, which is indeed incompatible with the view that the enzyme behaves as a dimer of dimers. This conclusion is confirmed by direct analysis of the rate curve. On the basis of the sum of the residuals, their sum of squares, the standard error of the kinetic parameters of the equation, the kinetic scheme associated with a dimer of dimers may be ruled out. On the basis of the same criteria, the fit of an Adair equation to the rate data cannot be retained as satisfactory. This is a direct proof that neither the Monod nor the Koshland model can correctly fit these kinetic data. In fact the model that fits these data best is a structural kinetic scheme where information transfer occurs between each subunit and its three neighbors ('tetrahedral' mode of information transfer). The fit of these models to a large number of kinetic data allows one to compute the free energy profile during the successive binding processes of the four substrate molecules to the enzyme. Whereas the first two steps are associated with an increase of free energy, all the other subsequent steps are associated with a decrease of free energy.

Chloroplasts↗

PubMed1998

The effects of age on kinetics of oxygen uptake and phosphocreatine in humans during exercise.

We compared the kinetics of oxygen uptake (VO2) and phosphocreatine (PCr) during the adjustment to and recovery from plantar flexion exercise in moderately active older (n = 10, 66.9 years) and younger (n = 10, 27.5 years) individuals. VO2 kinetics were similar in the two groups, with time constants (tau) averaging 46.3 +/- 10.2 s (younger, on-transient), 38.1 +/- 14.4 s (younger, off-transient), 46.3 +/- 17.8 (older, on-transient) and 40.7 +/- 19.2 s (older, off-transient). These were similar to corresponding PCr kinetics, measured by 31P nuclear magnetic resonance spectroscopy, which averaged 50.6 +/- 24.0 s (younger, on-transient), 42.0 +/- 16.1 s (younger, off-transient), 39.8 +/- 22.0 s (older, on-transient) and 37.6 +/- 21.6 s (older, off-transient). On-transient tau values for VO2 and PCr were correlated, for combined groups (r = 0.53; P = 0.015). We conclude that: (1) VO2 and PCr kinetics during exercise of a muscle group accustomed to daily activity are not compromised in physically active older humans, and (2) PCr kinetics reflect the kinetics of muscle O2 consumption, and are expressed at the lung (VO2 kinetics) after a transit delay.

Adult↗

PubMed1981

A kinetic analysis of the effects of adrenaline on calcium distribution in isolated rat liver parenchymal cells.

1. The effects of adrenaline on Ca distribution in isolated rat liver parenchymal cells were studied using a (45)Ca exchange technique under steady-state conditions with respect to the net movement of Ca. (45)Ca was initially introduced into the extracellular medium. The amount of cellular (45)Ca was determined after separation of the cells from the medium by centrifugation through a solution which contained LaCl(3) (to displace (45)Ca bound to sites on the outside of the cell membrane) and silicon oil. At 1.3 and 2.4 mm-extracellular Ca, a stimulation of the initial rate of (45)Ca exchange was observed in the presence of 10(-7)m-adrenaline (or 10(-6)m-phenylephrine) with a 7% decrease, and no change, respectively, in the plateau of the exchange curve. The same degree of stimulation was observed when (45)Ca was added at 1, 15, 30 or 45 min after the adrenaline.2. No stimulation of the initial rate of exchange was observed at 0.1 mm-extracellular Ca, or at 2.4 mm-extracellular Ca in the presence of antimycin A and oligomycin. At 0.1 mm-Ca, a 60% decrease in the plateau of the exchange curve was observed in the presence of adrenaline. The concentration of adrenaline (10(-7)m) which caused half-maximal stimulation of the initial rate of (45)Ca exchange at 1.3 mm-Ca was similar to that (2 x 10(-7)m) which caused half-maximal decrease in the plateau at 0.1 mm-Ca.3. The addition of adrenaline to cells equilibrated with (45)Ca at either 2.4 or 1.3 mm-Ca caused a transient loss of (45)Ca followed by a return to a new steady state after 1 or 10 min, respectively. A loss of (45)Ca was also observed at 0.1 mm-Ca, but the (45)Ca content of the cells remained maximally depressed for at least 30 min.4. A non-linear least-squares iterative curve-fitting technique was used to demonstrate that (a) an equation which includes two exponential terms and (b) a parallel or series arrangement of three compartments of exchangeable Ca (the medium and two compartments associated with the cell) are consistent with each set of data obtained at 1.3 or 2.4 mm-Ca in the presence or absence of adrenaline (or phenylephrine). At 1.3 mm-Ca, the quantities of exchangeable Ca in the two kinetically defined cellular compartments were 0.04-0.07 and 0.34-0.37 nmol per mg wet weight with rate constants for Ca outflow of 1.2-1.5 and 0.06-0.08 min(-1), respectively.5. Analysis of the changes induced by adrenaline or phenylephrine showed that at 1.3 and 2.4 mm-extracellular Ca these agents caused a 75-150% increase in the quantity of exchangeable Ca in the small kinetically defined compartment and a 20% decrease in the quantity of exchangeable Ca in the large kinetically defined compartment. These changes were mediated by an 80-160% increase in the rate constant for the inflow of Ca from the medium to the small kinetically defined compartment, and either a 20-60% decrease in the rate constant for inflow to, or a 20% increase in the rate constant for outflow from, the large compartment.6. Replacement of the LaCl(3) in the solution used to separate the cells from the incubation medium with either 5 mm-EGTA or 5 mm-CaCl(2) did not alter the kinetics of (45)Ca exchange or the stimulation by adrenaline. This, together with the observation that at 1.3 mm-extracellular Ca, adrenaline increases the initial rate of exchange in the absence, but not in the presence, of antimycin A plus oligomycin, indicates that both cellular compartments of exchangeable Ca are intracellular.7. The addition of antimycin A plus oligomycin to cells equilibrated with (45)Ca at 2.4 mm-extracellular Ca in the presence or absence of adrenaline displaced 0.09 and 0.14 nmol (45)Ca. mg(-1), respectively.8. Subcellular fractionation of cells equilibrated with (45)Ca at 0.1 mm-extracellular Ca revealed that the mitochondria and microsomes contained significant amounts of (45)Ca. The amounts of (45)Ca in these fractions decreased by 50 and 40%, respectively, in the presence of adrenaline.9. In (45)Ca exchange experiments conducted with isolated mitochondria at 37 degrees C at 1.5 x 10(-7)m and 0.9 x 10(-7)m free Ca in the presence of 2 mm-Mg(2+), one kinetically defined compartment of exchangeable mitochondrial Ca was detected. The rate constants for Ca outflow were found to be 0.15+/-0.03 and 0.12+/-0.04 min(-1), respectively, in reasonable agreement with the value obtained for the rate constant for the outflow of Ca from the large kinetically defined compartment of exchangeable Ca observed in cells.10. It is concluded that adrenaline has two effects on Ca movement in the liver cell. These are to cause a loss of Ca from an intracellular compartment, which includes the mitochondria and microsomes, and to increase the transport of Ca from the extracellular medium to an intracellular site. This results in an increase in the amount of Ca in a small intracellular compartment which may represent cytoplasmic Ca, or Ca bound to sites on the inside of the plasma membrane.

Animals↗

PubMed1986

'Concentration-clamp' study of gamma-aminobutyric-acid-induced chloride current kinetics in frog sensory neurones.

Kinetics of the activation and desensitization phases of gamma-aminobutyric acid (GABA)-induced Cl- current (ICl) were studied in single frog sensory neurones using the 'concentration-clamp' technique which enables perfusion of drugs with the time constant of about 3 ms. Both activation and desensitization phases of GABA response consisted of a single exponential at low concentrations and a double exponential at high concentrations. The time constant of the fast kinetic component in each phase was relatively stable, about 5 ms for activation and 3 s for desensitization over concentrations from 3 X 10(-5) to 3 X 10(-4) M, whereas those of the slow kinetic component decreased with increasing concentrations. The two kinetic components in both phases showed the same reversal potential. The slow and fast activation components recovered sensitivity from desensitization with different time courses: the recovery rate of the fast activation component was slow and that of the slow one, rapid. The peak ICl elicited at GABA concentrations below 10(-5) M increased disproportionally at more negative membrane potentials, thereby suggesting that the activation kinetics is voltage dependent. The steady-state ICl-voltage relationship obtained with less than 10(-5) M-GABA showed a non-linearity, probably due to voltage dependence of activation rather than that of desensitization kinetics. These results suggest the presence of at least two different GABA receptor-Cl- ionophore complexes with a different affinity and kinetics.

Action Potentials↗

PubMed2002

Polymorphic variants (CYP2C93 and CYP2C95) and the F114L active site mutation of CYP2C9: effect on atypical kinetic metabolism profiles.

CYP2C9 wild-type protein has been shown to exhibit atypical kinetic profiles of metabolism that may affect in vitro-in vivo predictions made during the drug development process. Previous work suggests a substrate-dependent effect of polymorphic variants of CYP2C9 on the rate of metabolism; however, it is hypothesized that these active site amino acid changes will affect the kinetic profile of a drug's metabolism as well. To this end, the kinetic profiles of three model CYP2C9 substrates (flurbiprofen, naproxen, and piroxicam) were studied using purified CYP2C9*1 (wild-type) and variants involving active site amino acid changes, including the naturally occurring variants CYP2C9*3 (Leu359) and CYP2C9*5 (Glu360) and the man-made mutant CYP2C9 F114L. CYP2C9*1 (wild-type) metabolized each of the three compounds with a distinctive profile reflective of typical hyperbolic (flurbiprofen), biphasic (naproxen), and substrate inhibition (piroxicam) kinetics. CYP2C9*3 metabolism was again hyperbolic for flurbiprofen, of a linear form for naproxen (no saturation noted), and exhibited substrate inhibition with piroxicam. CYP2C9*5-mediated metabolism was hyperbolic for flurbiprofen and piroxicam but linear with respect to naproxen turnover. The F114L mutant exhibited a hyperbolic kinetic profile for flurbiprofen metabolism, a linear profile for naproxen metabolism, and a substrate inhibition kinetic profile for piroxicam metabolism. In all cases except F114L-mediated piroxicam metabolism, turnover decreased and the K(m) generally increased for each allelic variant compared with wild-type enzyme. It seems that the kinetic profile of CYP2C9-mediated metabolism is dependent on both substrate and the CYP2C9 allelic variant, thus having potential ramifications on drug disposition predictions made during the development process.

Alleles↗

PubMed2003

The effect of incubation conditions on the enzyme kinetics of udp-glucuronosyltransferases.

Traditionally, the Michaelis-Menten equation has been used to determine kinetic parameters for in vitro glucuronidation assays. Recently, estradiol-3-glucuronide formation was shown to exhibit non-Michaelis-Menten kinetics consistent with autoactivation. A concern with the observation of nontraditional kinetics is that they may result as an artifact of the incubation conditions. To examine this concern, the formation of estradiol-3-glucuronide was investigated using human liver microsomes prepared by two different methods, a range of assay conditions, and activation by alamethecin, sonication, or Brij 58 (polyoxyethylene monocetyl ether). Interestingly, holding the other assay components constant, estradiol-3-glucuronide formation was up to 2.5-fold greater using microsomes prepared in phosphate buffer compared with those prepared in sucrose. Incubations activated by alamethecin consistently exhibited the highest rates of estradiol glucuronidation versus the other activators. Furthermore, estradiol-3-glucuronidation exhibited autoactivation kinetics in all of the conditions investigated (n = 1.2-1.7). Nontraditional kinetics were also observed when other UGT1A1 substrates such as ethinylestradiol, buprenorphine, and anthraflavic acid were studied with both human liver microsomes and recombinant UGT1A1. Naphthol, propofol, morphine, and androstanediol were used as probe UGT substrates selective for UGT1A6, UGT1A9, UGT2B7, and UGT2B15, respectively. Of these substrates, only androstanediol exhibited nontraditional kinetics using human liver microsomes. In conclusion, the Hill and/or Michaelis-Menten equations should be used to fit kinetic data to obtain an accurate assessment of in vitro glucuronidation.

Alamethicin↗

PubMed1985

Models for the kinetics of biodegradation of organic compounds not supporting growth.

We developed 12 models of kinetics to describe the metabolism of organic substrates that are not supporting bacterial growth. These models can be used to describe the biodegradation of organic compounds that are not supporting growth when the responsible populations are growing logistically, logarithmically, or linearly or are not increasing in numbers. Nonlinear regression analysis was used to fit patterns of mineralization by two bacteria to these kinetic models. Pseudomonas acidovorans mineralized 1 ng of phenol per ml while growing exponentially at the expense of uncharacterized organic carbon in a synthetic medium. Phenol at a concentration of 1 ng/ml did not affect the growth of P. acidovorans. These data were best fit by the model that incorporates the equation for logarithmic growth and assumes a concentration of test substrate well below its Km value. In the absence of a second substrate, glucose at concentrations below those supporting growth was mineralized by Salmonella typhimurium in a manner best described by pseudo first-order kinetics. In the presence of different concentrations of arabinose, however, the kinetics of glucose mineralization by S. typhimurium reflected linear, logistic, or logarithmic growth of the population on arabinose. We conclude that the kinetics of mineralization of organic compounds at concentrations too low to support growth are best described either by the first-order model or by models that incorporate expressions for the kinetics of growth of the metabolizing population on other substrates. When growth is at the expense of other substrates, the kinetics observed reflect such growth, as well as the concentration of the substrate of interest.(ABSTRACT TRUNCATED AT 250 WORDS)

Arabinose↗

PubMed1995

In vivo measurements of pulmonary angiotensin-converting enzyme kinetics. I. Theory and error analysis.

We developed a procedure for measuring pulmonary angiotensin-converting enzyme kinetics with fluorine-18 fluorocaptopril and positron emission tomography (PET). The method is based on the application of a compartmental receptor model that represents the kinetics of two species of ligand, presumably the trans and cis conformers of captopril. The input function was characterized and includes corrections for the labeled metabolites of fluorocaptopril. Application of the procedure to lung time-activity data obtained with PET produced estimates of kinetic parameters demonstrating fast kinetics for one conformer and slower kinetics for the other. Simulation studies were performed to evaluate the sensitivity of the estimated parameters to errors in the model assumptions and in measured values for variables required for analysis of the PET data. Estimates for two of the kinetic parameters, the amount of perfused unbound functional enzyme normalized to regional lung volume and the association rate constant for the trans conformer, were relatively stable even with large errors in the input data, varying < 30% from true values for all perturbations. Thus, the procedure produces reliable estimates of the kinetics of the trans conformer of captopril as well as theoretical curves that are close to the observed data.

Animals↗

PubMed1997

Gas exchange and cardiovascular kinetics with different exercise protocols in heart transplant recipients.

Metabolic and cardiovascular adjustments to various submaximal exercises were evaluated in 82 heart transplant recipients (HTR) and in 35 control subjects (C). HTR were tested 21.5 +/- 25.3 (SD) mo (range 1.0-137.1 mo) posttransplantation. Three protocols were used: protocol A consisted of 5 min of rectangular 50-W load repeated twice, 5 min apart [5 min rest, 5 min 50 W (Ex 1), 5 min recovery, 5 min 50 W (Ex 2)]; protocol B consisted of 5 min of rectangular load at 25, 50, or 75 W; protocol C consisted of 15 min of rectangular load at 25 W. Breath-by-breath pulmonary ventilation (VE), O2 uptake (VO2), and CO2 output (VCO2) were determined. During protocol A, beat-by-beat cardiac output (Q) was estimated by impedance cardiography. The half times (t1/2) of the on- and off-kinetics of the variables were calculated. In all protocols, t1/2 values for VO2 on-, VE on-, and VCO2 on-kinetics were higher (i.e., the kinetics were slower) in HTR than in C, independently of workload and of the time post-transplantation. Also, t1/2 Q on- was higher in HTR than in C. In protocol A, no significant difference of t1/2 VO2 on- was observed in HTR between Ex 1 (48 +/- 9 s) and Ex 2 (46 +/- 8 s), whereas t1/2 Q on- was higher during Ex 1 (55 +/- 24 s) than during Ex 2 (47 +/- 15 s). In all protocols and for all variables, the t1/2 off-values were higher in HTR than in C, In protocol C, no differences of steady-state VE, VO2, and VCO2 were observed in both groups between 5, 10, and 15 min of exercise. We conclude that 1) in HTR, a "priming" exercise, while effective in speeding up the adjustment of convective O2 flow to muscle fibers during a second on-transition, did not affect the VO2 on-kinetics, suggesting that the slower VO2 on- in HTR was attributable to peripheral (muscular) factors; 2) the dissociation between Q on- and VO2 on-kinetics in HTR indicates that an inertia of muscle metabolic machinery is the main factor dictating the VO2 on-kinetics; and 3) the VO2 off-kinetics was slower in HTR than in C, indicating a greater alactic O2 deficit in HTR and, therefore, a sluggish muscle VO2 adjustment.

Adolescent↗

PubMed2004

Downhill kinetics of biomolecular interface binding: globally connected scenario.

We study the kinetics of the biomolecular binding process at the interface using energy landscape theory. The global kinetic connectivity case is considered for a downhill funneled energy landscape. By solving the kinetic master equation, the kinetic time for binding is obtained and shown to have a U-shape curve-dependence on the temperature. The kinetic minimum of the binding time monotonically decreases when the ratio of the underlying energy gap between native state and average non-native states versus the roughness or the fluctuations of the landscape increases. At intermediate temperatures, fluctuations measured by the higher moments of the binding time lead to non-Poissonian, non-exponential kinetics. At both high and very low temperatures, the kinetics is nearly Poissonian and exponential.

Binding Sites↗

PubMed2005

Equilibrium and kinetic folding pathways of a TIM barrel with a funneled energy landscape.

The role of native contact topology in the folding of a TIM barrel model based on the alpha-subunit of tryptophan synthase (alphaTS) from Salmonella typhimurium (Protein Data Bank structure 1BKS) was studied using both equilibrium and kinetic simulations. Equilibrium simulations of alphaTS reveal the population of two intermediate ensembles, I1 and I2, during unfolding/refolding at the folding temperature, Tf = 335 K. Equilibrium intermediate I1 demonstrates discrete structure in regions alpha0-beta6 whereas intermediate I2 is a loose ensemble of states with N-terminal structure varying from at least beta1-beta3 (denoted I2A) to alpha0-beta4 at most (denoted I2B). The structures of I1 and I2 match well with the two intermediate states detected in equilibrium folding experiments of Escherichia coli alphaTS. Kinetic folding simulations of alphaTS reveal the sequential population of four intermediate ensembles, I120Q, I200Q, I300Q, and I360Q, during refolding. Kinetic intermediates I120Q, I200Q, and I300Q are highly similar to equilibrium alphaTS intermediates I2A, I2B, and I1, respectively, consistent with kinetic experiments on alphaTS from E. coli. A small population (approximately 10%) of kinetic trajectories are trapped in the I120Q intermediate ensemble and require a slow and complete unfolding step to properly refold. Both the on-pathway and off-pathway I120Q intermediates show structure in beta1-beta3, which is also strikingly consistent with kinetic folding experiments of alphaTS. In the off-pathway intermediate I(120Q), helix alpha2 is wrapped in a nonnative chiral arrangement around strand beta3, sterically preventing the subsequent folding step between beta3 and beta4. These results demonstrate the success of combining kinetic and equilibrium simulations of minimalist protein models to explore TIM barrel folding and the folding of other large proteins.

Computer Simulation↗

PubMed2006

Sorption kinetics and equilibria of organic pesticides in carbonatic soils from South Florida.

A batch reactor was used to determine sorption kinetic parameters (k2, F, and K*) and the equilibrium sorption coefficient (K). The two-site nonequilibrium (TSNE) batch sorption kinetics model was used to calculate the kinetic parameters. Two probe organic pesticides, atrazine [2-chloro-4-ethylamino-6-isopropylamino-s-triazine] and diuron [3-(3,4-dichlorophenyl)-1,1-dimethylurea] were studied using three carbonatic soils from South Florida (Chekika, Perrine, and Krome), one noncarbonatic soil from Iowa (Webster), and one organic soil (Lauderhill) from South Florida. Carbonatic soils contained more than 600 g kg(-1) CaCO3. Sorption is initially very fast up to 3 h and then slowly reaches equilibrium. All soil-chemical combinations reached sorption equilibrium after about 24 h and all sorption isotherms were linear. The sorption kinetics data were well described by the TSNE model for all soil-chemical combinations except for the marl soil data (Perrine-Atrazine), which were better described by the one-site nonequilibrium (OSNE) model. Diuron, with higher K, undergoes slower sorption kinetics than atrazine. The Lauderhill soil containing organic carbon (OC) of 450 g kg(-1) exhibited slowest sorption kinetics for both pesticides. An inverse relationship between k3 and K was observed for atrazine and diuron separately in Chekika, Webster, and Lauderhill soils but not in Perrine and Krome soils. The sorption kinetic parameters were used to distinguish the sorption behavior between atrazine and diuron and to identify differences between soils. Normalizing the sorption coefficient (K) to OC showed that atrazine and diuron had K oc values in carbonatic soils that were a third of reported literature values for noncarbonatic soils. Using existing literature K oc values in solute transport models will most likely underestimate the mobility of atrazine, diuron, and other neutral organic chemicals in carbonatic soils.

Carbonates↗

PubMedDate not supplied

Drug kinetics and alcohol ingestion.

Acute and chronic ethanol ingestion can alter both the pharmacodynamics and pharmacokinetics of other drugs. For psychotherapeutic drugs, modification of drug action by alcohol is much more important than kinetic interaction, such as ethanol induced drug metabolism. In contrast, the importance of the effects of alcohol on the kinetics of other classes of drug is incomplete. The probability and mechanism of alcohol kinetic interactions with other drugs can nevertheless be anticipated, in part, on the basis of the extent of binding of the drug to plasma proteins, the capacity of the liver for extracting the drug from blood passing through the liver and the true distribution space of the drug. Highly bound drugs with low intrinsic hepatic clearance are among the most commonly reported to have their kinetics altered by ethanol (e.g. benzodiazepines, phenytoin, tolbutamide and warfarin). Less highly bound drugs are less consistently affected (e.g. meprobamate, glutethimide, pentobarbitone and phenobarbitone). Acute administration of ethanol to laboratory animals or incubation of microsomal preparations with ethanol inhibits the mixed function oxidase activity. In the human, the elimination half-life of meprobamate, pentobarbitone and tolbutamide is increased by acute ethanol administration. Chronic administration of ethanol to rats and humans causes proliferation of the smooth endoplasmic reticulum, increase in microsomal protein content and cytochrome P450 and results in an augmentation in drug metabolising ability of the microsomes in vitro. Even though the plasma half-life of some drugs is decreased by chronic ethanol ingestion, the clinical determination of the mechanism is incomplete because few studies have measured drug metabolite levels. In addition, alcohol effects on drug distribution have not been studied very extensively. The effects of chronic alcohol ingestion on drugs with low and high hepatic extraction, high and low binding, important tissue localisation and microsomal and non-microsomal metabolism will be quite different. Systematic studies of the mechanism of alcohol kinetic interactions are needed. Such kinetic studies should be combined with pharmacodynamic measures in order to establish the clinical importance of changes in drug kinetics.

Alcoholism↗

PubMed1995

Insulin sensitivity accounts for glucose and lactate kinetics after intravenous glucose injection.

Mathematical modeling was used to explore the interaction between glucose, insulin, and lactate during the frequently sampled intravenous glucose tolerance test (FSIGTT). Insulin-modified FSIGTs were performed in 25 lean volunteers, and an additional 5 volunteers underwent FSIGTs with glucose injection alone to illustrate the effect of insulin on both glucose and lactate kinetics. The model chosen as the best representation of the system extended the minimal model of glucose kinetics (MM) by including a two-compartment model of lactate kinetics. The model accounted for both glucose and lactate kinetics, provided traditional MM parameters of insulin sensitivity and glucose effectiveness, and descriptive parameters of lactate kinetics. Modeling suggested that lactate production was limited by the rate of glucose disappearance, with no indication of direct effects of insulin on lactate. Inclusion of lactate kinetics had no adverse effect on MM parameters (SG: 0.023 +/- 0.009 vs. 0.023 +/- 0.010 min-1, SI: 1.01 +/- 0.70 vs. 1.03 +/- 0.71 x 10(4).min-1.pmol-1.1; P > 0.50, lactate model vs. MM), and indicated that approximately 1.2% min-1 of total glucose disappearance during the FSIGT is converted to lactate. An additional benefit of including lactate kinetics was the significant improvement in precision in MM parameter estimates as reflected by the fractional standard deviations (FSDs). This effect was most prominent for SG, in which a threefold improvement in parameter precision was observed (FSD: 13.5 +/- 3.1 vs. 42.5 +/- 48.5; means +/- SD).(ABSTRACT TRUNCATED AT 250 WORDS)

Adult↗

PubMed2000

Mechanisms of calcium decay kinetics in hippocampal spines: role of spine calcium pumps and calcium diffusion through the spine neck in biochemical compartmentalization.

Dendritic spines receive most excitatory inputs in the CNS and compartmentalize calcium. Although the mechanisms of calcium influx into spines have been explored, it is unknown what determines the calcium decay kinetics in spines. With two-photon microscopy we investigate action potential-induced calcium dynamics in spines from rat CA1 pyramidal neurons in slices. The [Ca(2+)](i) in most spines shows two decay kinetics: an initial fast component, during which [Ca(2+)](i) in spines decays to dendritic levels, followed by a slower decay phase in which the spine follows dendritic kinetics. The correlation between [Ca(2+)](i) in spine and dendrite at the breakpoint of the decay kinetics demonstrates diffusional equilibration between spine and dendrite during the slower component. To explain the faster initial decay, we rule out saturation or kinetic effects of endogenous or exogenous buffers and focus instead on (1) active calcium extrusion and (2) buffered diffusion of calcium from spine to dendrite. The presence of an undershoot in most spines indicates that extrusion mechanisms can be intrinsic to the spine. Supporting the two mechanisms, pharmacological blockade of smooth endoplasmic reticulum calcium (SERCA) pumps and the length of the spine neck affect spine decay kinetics. Using a mathematical model, we find that the contribution of calcium pumps and diffusion varies from spine to spine. We conclude that dendritic spines have calcium pumps and that their density and kinetics, together with the morphology of the spine neck, determine the time during which the spine compartmentalizes calcium.

Action Potentials↗