Pharmacokinetics/pharmacodynamics during phase II and III studies: population kinetics and the kinetic screen.
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Clinical Trials, Phase II as Topic↗
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The test-retest reliability of gas exchange kinetics in humans using a pseudo random binary sequence exercise test.
The purpose of this study was to compare the test-retest reliability of oxygen uptake (VO2) kinetics with carbon dioxide output (VCO2) kinetics using a pseudo random binary sequence (PRBS) exercise test. A reliable test of gas exchange kinetics would have the potential of being applied as a sports fitness test. Ten healthy male subjects agreed to participate in the study and all subjects completed two identical PRBS exercise tests (test 1 and test 2), separated by a 30 min period of inactivity. Three consecutive 300 s PRBS cycles were completed in each test with 20 s exercise intensity changes between 25 and 85 W using an electrically braked cycle ergometer. Fourier analysis was computed for frequencies 3.3, 6.7 and 10 mHz. Statistical analysis by two-way ANOVA with repeated measures did not reveal significant differences between test 1 and test 2 for either VO2 kinetics or VCO2 kinetics. Static gain of VO2 for test 1 [9.11 (SD 0.59) ml.min-1.W-1] and test 2 [9.23 (SD 0.64) ml.min-1.W-1] did not differ significantly between tests. The 95% limits of agreement for VCO2 kinetics displayed increased variability in comparison to VO2 kinetics at each frequency of amplitude ratio and phase shift. Systematic bias ranged between 0% and 4%, except at frequency 10 mHz of VCO2 kinetics phase shift which showed a 10% bias for slower VO2 kinetics in test 2. It is possible that the increased variability of VCO2 kinetics compared to VO2 kinetics might be attributable to a lower signal to noise ratio in VCO2 kinetics, variations in ventilation or the storage mechanisms of CO2. The lower variability of VO2 kinetics compared with VCO2 kinetics suggests that the PRBS test of VO2 kinetics has the greater potential for further development as an indicator of aerobic fitness.
Adult↗
k-Cone analysis: determining all candidate values for kinetic parameters on a network scale.
The absence of comprehensive measured kinetic values and the observed inconsistency in the available in vitro kinetic data has hindered the formulation of network-scale kinetic models of biochemical reaction networks. To meet this challenge we present an approach to construct a convex space, termed the k-cone, which contains all the allowable numerical values of the kinetic constants in large-scale biochemical networks. The definition of the k-cone relies on the incorporation of in vivo concentration data and a simplified approach to represent enzyme kinetics within an established constraint-based modeling approach. The k-cone approach was implemented to define the allowable combination of numerical values for a full kinetic model of human red blood cell metabolism and to study its correlated kinetic parameters. The k-cone approach can be used to determine consistency between in vitro measured kinetic values and in vivo concentration and flux measurements when used in a network-scale kinetic model. k-Cone analysis was successful in determining whether in vitro measured kinetic values used in the reconstruction of a kinetic-based model of Saccharomyces cerevisiae central metabolism could reproduce in vivo measurements. Further, the k-cone can be used to determine which numerical values of in vitro measured parameters are required to be changed in a kinetic model if in vivo measured values are not reproduced. k-Cone analysis could identify what minimum number of in vitro determined kinetic parameters needed to be adjusted in the S. cerevisiae model to be consistent with the in vivo data. Applying the k-cone analysis a priori to kinetic model development may reduce the time and effort involved in model building and parameter adjustment. With the recent developments in high-throughput profiling of metabolite concentrations at a whole-cell scale and advances in metabolomics technologies, the k-cone approach presented here may hold the promise for kinetic characterization of metabolic networks as well as other biological functions at a whole-cell level.
Algorithms↗
Development of fractal kinetic theory for enzyme-catalysed reactions and implications for the design of biochemical pathways.
Recent evidence has shown that elementary bimolecular reactions under dimensionally-restricted conditions, such as those that might occur within cells when reactions are confined to two-dimensional membranes and one-dimensional channels, do not follow traditional mass-action kinetics, but fractal kinetics. The power-law formalism, which provides the context for examining the kinetics under these conditions, is used here to examine the implications of fractal kinetics in a simple pathway of reversible reactions. Starting with elementary chemical kinetics, we proceed to characterise the equilibrium behaviour of a simple bimolecular reaction, derive a generalised set of conditions for microscopic reversibility, and develop the fractal kinetic rate law for a reversible Michaelis-Menten mechanism. Having established this fractal kinetic framework, we go on to analyse the steady-state behaviour and temporal response of a pathway characterised by both the fundamental and quasi-steady-state equations. These results are contrasted with those for the fundamental and quasi-steady-state equations based on traditional mass-action kinetics. Finally, we compare the accuracy of three local representations based on both fractal and mass-action kinetics. The results with fractal kinetics show that the equilibrium ratio is a function of the amount of material in a closed system, and that the principle of microscopic reversibility has a more general manifestation that imposes new constraints on the set of fractal kinetic orders. Fractal kinetics in a biochemical pathway allow an increase in flux to occur with less accumulation of pathway intermediates and a faster temporal response than is the case with traditional kinetics. These conclusions are obtained regardless of the level of representation considered. Thus, fractal kinetics provide a novel means to achieve important features of pathway design.
Catalysis↗
Atypical cytochrome p450 kinetics: implications for drug discovery.
The Michaelis-Menten model is commonly used to estimate a drug's potential in vivo hepatic clearance based on in vitro data obtained during drug discovery and development. This paradigm assumes that the drug obeys 'typical' enzyme kinetics and thus can be described by this model. However, it is increasingly being recognised that a number of drugs metabolised not only by the cytochrome P450 enzymes but also by other enzymes and transporters can exhibit atypical kinetic profiles, and thus are not accurately modeled with the Michaelis-Menten model. Application of an incorrect model can then lead to mis-estimation of in vitro intrinsic clearance and thus affect the prediction of in vivo clearance. This review discusses several types of atypical kinetic profiles that may be observed, including examples of homotropic cooperativity (i.e. sigmoidal kinetics, biphasic kinetics and substrate inhibition kinetics) as well as heterotropic cooperativity (i.e. activation). Application of the incorrect kinetic model may profoundly affect estimations of intrinsic clearance. For example, incorrectly applying the Michaelis-Menten model to a kinetic profile exhibiting substrate inhibition kinetics will result in an underestimation of Km (Michaelis-Menten constant) and V(max) (maximal velocity), whereas application of the Michaelis-Menten model to sigmoidal kinetic data typically results in an overestimation of Km and V(max) at the lower substrate concentrations that are typically therapeutically relevant. One must also be careful of potential artefactual causes of atypical kinetic profiles, such as enzyme activation by solvents, buffer dependent kinetic profiles, or altered kinetic parameter estimates due to nonspecific binding of the substrate to proteins. Despite a plethora of data on the effects of atypical kinetic profiles in vitro, only modest effects have been noted in vivo (with the exception of substrate dependent inhibition). Thus, the clinical relevance of these phenomena remains inconclusive.
Algorithms↗
Enzyme kinetics of cytochrome P450-mediated reactions.
The most common drug-drug interactions may be understood in terms of alterations of metabolism, associated primarily with changes in the activity of cytochrome P450 (CYP) enzymes. Kinetic parameters such as Km, Vmax, Ki and Ka, which describe metabolism-based drug interactions, are usually determined by appropriate kinetic models and may be used to predict the pharmacokinetic consequences of exposure to one or multiple drugs. According to classic Michaelis-Menten (M-M) kinetics, one binding site models can be employed to simply interpret inhibition (pure competitive, non-competitive and uncompetitive) or activation of the enzyme. However, some cytochromes P450, in particular CYP3A4, exhibit unusual kinetic characteristics. In this instance, the changes in apparent kinetic constants in the presence of inhibitor or activator or second substrate do not obey the rules of M-M kinetics, and the resulting kinetics are not straightforward and hamper mechanistic interpretation of the interaction in question. These unusual kinetics include substrate activation (autoactivation), substrate inhibition, partial inhibition, activation, differential kinetics and others. To address this problem, several kinetic models can be proposed, based upon the assumption that multiple substrate binding sites exist at the active site of a particular P450, and the resulting kinetic constants are, therefore, solved to adequately describe the observed interaction between multiple drugs. The following is an overview of some cytochrome P450-mediated classic and atypical enzyme kinetics, and the associated kinetic models. Applications of these kinetic models can provide some new insights into the mechanism of P450-mediated drug-drug interactions.
Algorithms↗
Maximum entropy, analysis of kinetic processes involving chemical and folding-unfolding changes in proteins.
We show that numerical inversion of the Laplace transform by using the maximum entropy method can be successfully applied to the analysis of complex kinetic processes involving chemical and folding-unfolding changes in proteins. First, we present analyses of simulated data which support that: (i) the maximum entropy calculation of rate distributions, combined with Monte Carlo analyses of the associated uncertainties, yields results consistent with the information actually supplied by the data, thus preventing their over-interpretation; (ii) maximum entropy analysis may be used to extract discrete rates (corresponding to individual exponential contributions), as well as broad rate distributions (provided, of course, that the adequate information is supplied by the data). We further illustrate the applicability of the maximum entropy analysis with experimental data corresponding to two nontrivial model processes: (a) the kinetics of chemical modification of sulfhydryl groups in glycogen synthase by reaction with Ellman's reagent; (b) the kinetics of folding of ribonuclease a under strongly folding conditions, as monitored by fluorescence and optical absorption. Finally, we discuss that the maximum entropy approach should be particularly useful in studies on protein folding kinetics, which generally involve the comparison between several complex kinetic profiles obtained by using different physical probes. Thus, protein folding kinetics is usually interpreted in terms of kinetic mechanisms involving a comparatively small number of kinetic steps between well-defined protein states. According to this picture, rate distributions derived from experimental kinetic profiles by maximum entropy analysis are expected to show a small number of comparatively narrow peaks, from which we can determine, without a priori assumptions, the number of exponential contributions required to describe each experimental kinetic profile (the number of peaks), together with their amplitudes (from the peak areas), time constant values (from the peak positions), and associated Monte Carlo uncertainties. On the other hand, recent theoretical studies describe protein folding kinetics in terms of the protein energy landscape (the multidimensional surface of energy versus conformational degrees of freedom), emphasize the difficulty in defining a single reaction coordinate for folding, and point out that individual chains may fold by multiple pathways. This indicates that, in some cases at least, folding kinetics might have to be described in terms of broad rate distributions (rather than in terms of a small number of discrete exponential contributions related to kinetic steps between well-defined protein states). We suggest that the maximum entropy procedures described in this work may provide a method to detect this situation and to derive such broad rate distributions from experimental data.
Entropy↗
Kinetics of protein adsorption and desorption on surfaces with grafted polymers.
The kinetics of protein adsorption are studied using a generalized diffusion approach which shows that the time-determining step in the adsorption is the crossing of the kinetic barrier presented by the polymers and already adsorbed proteins. The potential of mean-force between the adsorbing protein and the polymer-protein surface changes as a function of time due to the deformation of the polymer layers as the proteins adsorb. Furthermore, the range and strength of the repulsive interaction felt by the approaching proteins increases with grafted polymer molecular weight and surface coverage. The effect of molecular weight on the kinetics is very complex and different than its role on the equilibrium adsorption isotherms. The very large kinetic barriers make the timescale for the adsorption process very long and the computational effort increases with time, thus, an approximate kinetic approach is developed. The kinetic theory is based on the knowledge that the time-determining step is crossing the potential-of-mean-force barrier. Kinetic equations for two states (adsorbed and bulk) are written where the kinetic coefficients are the product of the Boltzmann factor for the free energy of adsorption (desorption) multiplied by a preexponential factor determined from a Kramers-like theory. The predictions from the kinetic approach are in excellent quantitative agreement with the full diffusion equation solutions demonstrating that the two most important physical processes are the crossing of the barrier and the changes in the barrier with time due to the deformation of the polymer layer as the proteins adsorb/desorb. The kinetic coefficients can be calculated a priori allowing for systematic calculations over very long timescales. It is found that, in many cases where the equilibrium adsorption shows a finite value, the kinetics of the process is so slow that the experimental system will show no adsorption. This effect is particularly important at high grafted polymer surface coverage. The construction of guidelines for molecular weight/surface coverage necessary for kinetic prevention of protein adsorption in a desired timescale is shown. The time-dependent desorption is also studied by modeling how adsorbed proteins leave the surface when in contact with a pure water solution. It is found that the kinetics of desorption are very slow and depend in a nonmonotonic way in the polymer chain length. When the polymer layer thickness is shorter than the size of the protein, increasing polymer chain length, at fixed surface coverage, makes the desorption process faster. For polymer layers with thickness larger than the protein size, increases in molecular weight results in a longer time for desorption. This is due to the grafted polymers trapping the adsorbed proteins and slowing down the desorption process. These results offer a possible explanation to some experimental data on adsorption. Limitations and extension of the developed approaches for practical applications are discussed.
Adsorption↗
Modeling kinetic data from in vitro drug metabolism enzyme experiments.
Modeling of in vitro enzyme kinetic data derived from drug metabolism experiments can greatly facilitate the drug development process because estimation of kinetic parameters can facilitate decision making regarding whether to continue development of a compound. From this information, predictions can be made regarding the "metabolic stability" of a compound and even the in vivo intrinsic clearance of the drug. Many drugs exhibit typical Michaelis-Menten-type kinetics in vitro that result in a hyperbolic kinetic profile from which Km and Vm can be readily estimated. However, it is increasingly being recognized that many drug compounds exhibit "atypical" enzyme kinetics in vitro, requiring use of more complex kinetic models for data fitting and parameter estimation. These atypical kinetic profiles may include sigmoidal kinetics (autoactivation), biphasic kinetics, substrate inhibition kinetics, and heterotropic cooperativity (activation). This article briefly summarizes the types of equations necessary to adequately model both typical and atypical kinetic profiles in order to facilitate correct estimation of the relevant kinetic parameters.
Algorithms↗
Kinetics of estimated human muscle capillary blood flow during recovery from exercise.
The kinetic characteristics of muscle capillary blood flow (Qcap) during recovery from exercise are controversial (e.g. one versus two phases). Furthermore, it is not clear how the overall Qcap kinetics are temporally associated with muscle oxygen uptake (VO2m) kinetics. To address these issues, we examined the kinetics of Qcap estimated from the rearrangement of the Fick equation (Qcap=VO2m/C(a-v)O2) using the kinetics of pulmonary VO2 (VO2p, primary component) and deoxy-haemoglobin concentration ([HHb]) as indices of VO2m and C(a - v)O2 (arterio-venous oxygen difference) kinetics, respectively. VO2p (l min-1) was measured breath by breath and [HHb] (microm) was measured by near infrared spectroscopy during moderate (M; below lactate threshold, LT) and heavy exercise (H, above LT) in nine subjects. The kinetics of Qcap were biphasic, with an initial fast phase (tauI; M=9.3+/-4.9 s and H=6.0+/-3.8 s) followed by a slower phase 2 (tauP; M=29.9+/-8.6 s and H=47.7+/-26.0 s). For moderate exercise, the overall kinetics of Qcap (mean response time [MRT], 36.1+/-8.6 s) were significantly slower than the kinetics of VO2p (tauP; 27.8+/-5.3 s) and [HHb] (MRT for [HHb]; 16.2+/-6.3 s). However, for heavy exercise, there was no significant difference between MRT-[HHb] (34.7+/-10.4 s) and tauP for VO2p (32.3+/-6.7 s), while MRT for Qcap (48.7+/-21.8 s) was significantly slower than MRT for [HHb] and tauP for VO2p. In conclusion, during recovery from exercise the estimated Qcap kinetics were biphasic, showing an early rapid decrease in blood flow. In addition, the overall kinetics of Qcap were slower than the estimated VO2m kinetics.
Adult↗
Processing of kinetically defined boundaries in the cortical motion area MT of the macaque monkey.
1. Electrophysiological recordings of 68 cells in the middle temporal area MT were made in paralyzed and anesthetized macaque monkeys. 2. Testing with our kinetic boundary stimuli always occurred under optimized conditions. To this end, the preferred direction, speed, stimulus position, and stimulus size of each cell were determined by quantitative tests. 3. The orientation selectivity to stationary luminance contrast edges served as a reference by which a response to kinetic boundaries could be compared. We found cells in area MT to be less selective to the orientation of luminance contrast stimuli than to the direction of motion. We confirmed the presence of neurons with preferred orientation aligned with their preferred direction. 4. The responses to kinetic edges defined by motion vectors moving in opposite directions, kinetic gratings with motion vectors in opposite directions, kinetic edges containing coherent motion and a stationary complementary field or coherent motion and a complementary field containing visual dynamic noise were compared. Kinetic boundaries were generated so that the motion vectors moved either parallel or orthogonal to the orientation of the discontinuity. For a cell to be considered as responding to the orientation of a kinetic boundary, it had to exhibit the same preferred orientation when the local motion vectors changed from parallel to orthogonal to the orientation of the kinetic boundary. 5. All cells in area MT changed their preferred orientation by 90 degrees when the coherent motion vectors changed from moving parallel to moving orthogonal to the boundary. This was the case independent of the types of kinetic boundary tested. We concluded that cells in area MT appear to respond to the motion vector over their classical receptive field (CRF) only and were unable to code the orientation of the kinetic boundary. 6. In those cells exhibiting an antagonistic surround, we examined the ability of the cell to code the position of a kinetic boundary. None of the cells tested signaled the position of a kinetic boundary. The side preference of the stimulus of the cells changed from left to right as the motion vectors in the stimulus reversed. This indicates that the cells were only selective for the motion vectors present over their CRF. 7. We found that the directional sensitivity of cells in area MT remained unaltered by the presence of additional motion vectors within the CRF. This suggests that cells in area MT extract a specific motion vector from a spatial configuration of vectors.(ABSTRACT TRUNCATED AT 400 WORDS)
Animals↗
Probing the kinetics of single molecule protein folding.
We propose an approach to integrate the theory, simulations, and experiments in protein-folding kinetics. This is realized by measuring the mean and high-order moments of the first-passage time and its associated distribution. The full kinetics is revealed in the current theoretical framework through these measurements. In the experiments, information about the statistical properties of first-passage times can be obtained from the kinetic folding trajectories of single molecule experiments (for example, fluorescence). Theoretical/simulation and experimental approaches can be directly related. We study in particular the temperature-varying kinetics to probe the underlying structure of the folding energy landscape. At high temperatures, exponential kinetics is observed; there are multiple parallel kinetic paths leading to the native state. At intermediate temperatures, nonexponential kinetics appears, revealing the nature of the distribution of local traps on the landscape and, as a result, discrete kinetic paths emerge. At very low temperatures, exponential kinetics is again observed; the dynamics on the underlying landscape is dominated by a single barrier. The ratio between first-passage-time moments is proposed to be a good variable to quantitatively probe these kinetic changes. The temperature-dependent kinetics is consistent with the strange kinetics found in folding dynamics experiments. The potential applications of the current results to single-molecule protein folding are discussed.
Computer Simulation↗
Light microscopy diagnosis of Babesia bovis and Babesia bigemina kinetes in the haemolymph of artificially infected Boophilus microplus engorged female ticks.
The length, width and position of the nucleus of Babesia bovis and Babesia bigemina kinetes from the haemolymph of Boophilus microplus engorged female ticks were recorded. Additionally, the shape of Babesia bovis kinetes were registered as curved, semi-curved or straight. To this aim Boophilus microplus tick larvae from a colony free of Babesia were fed on splenectomised calves artificially infected with either Babesia bovis or Babesia bigemina pathogenic strains. Six engorged female ticks showing an infection of at least ten mature kinetes of Babesia bovis in a sample of haemolymph 5 days after detachment were also monitored 7, 9 and 10 days after collection. The same procedure was followed with six engorged female ticks infected with Babesia bigemina. One hundred and twenty kinetes of each species of Babesia were evaluated. The mean length +/- standard deviation and ranges for Babesia bovis kinetes were 14.30 +/- 0.922 microns and 11.9-16.3 microns, while the corresponding measures for the kinetes of Babesia bigemina were 11.27 +/- 0.900 microns and 9.0-13.1 microns (P < 0.001, t-test). The width was 3.33 +/- 0.315 microns, 2.6-4.0 microns for Babesia bovis and 2.24 +/- 0.287 microns, 1.5-2.8 microns for Babesia bigemina kinetes (P < 0.001). The most common position of the nucleus was central for both species of Babesia. A total of 58% of Babesia bovis kinetes showed the typical curved tail. No effect of time post-collection and individual host ticks in the kinete of Babesia bigemina was found while an unexpected influence of individual host tick in the width of Babesia bovis kinetes was detected (P < 0.01, analysis of variance). The overlap in the sizes of kinetes from both species of Babesia makes it difficult to apply the results to ticks of unknown babesial infection status. This finding is further complicated by the intra-specific size variations of Babesia kinetes from different geographical origins.
Animals↗
Kinetic models for insulin disappearance from plasma in man.
The general use of first order kinetics to describe the disappearance of insulin from plasma in man contrasts the available evidence of saturation kinetics for insulin. In order to bridge this gap, we have put forward three alternative models of insulin kinetics. Model 1 considers the combined existence of first order and saturation (Michaëlis-Menten) kinetics. Model 2 considers saturation kinetics alone. Model 3 considers first order kinetics alone. The validity of the models was studied in normal and type I (insulin-dependent) diabetic subjects. Sequential constant intravenous infusions of insulin at different rates were used to achieve different levels of steady state plasma insulin concentrations, while the glycaemic level (usually normoglycaemia) was maintained by a glucose clamp. Appropriate validation procedures demonstrated that the model of saturation kinetics alone (model 2) was superior to the other models in normal subjects at physiological and supraphysiological plasma insulin concentrations, and in diabetic patients at supraphysiological concentrations. The minimal model at physiological insulin concentrations in type I diabetic patients was that of first order kinetics (model 3). The kinetics of insulin was independent of the species of insulin (human or porcine) in both study groups. The actual glycaemic clamp level (normoglycaemia and moderate hyperglycaemia) did not influence the insulin disappearance rate. Binding of insulin to its receptor is considered to be the initial step in insulin degradation. Saturation kinetics of insulin may therefore be influenced by the saturation of binding of insulin molecules to their receptors. We found values of Km (i.e. the concentration of plasma insulin at which the insulin disappearance rate is half maximal) in normal subjects comparable to values of Kd (i.e. the dissociation constant for insulin-receptor binding) from receptor studies in isolated cells. Changes in regional (hepatic and/or renal) blood flow at hyperinsulinaemia represent an alternative explanation to a model of saturation kinetics. Increases in Vmax (i.e. the maximal insulin disappearance rate) and Km with increasing insulin dose were observed in normal subjects. This finding suggests that insulin may disappear from plasma by more than one saturable pathway. Additional studies are needed to confirm this observation. The clearance rate of insulin at infinitesimal plasma insulin concentrations (Vmax/Km) was 28 +/- 8 ml.kg-1.min-1 in normal subjects. This value is higher than most clearance rates previously reported in studies using first order kinetics. The clearance rate of insulin in type I diabetic patients was 20 +/- 4 ml.kg-1.min-1, corresponding to a reduction in clearance of 30% compared to normal subjects.(ABSTRACT TRUNCATED AT 400 WORDS)
Humans↗
Influence of fractal kinetics on molecular recognition.
Molecular recognition is a central issue for nearly every biological mechanism. The analysis of molecular recognition to date has been conducted within the framework of classical chemical kinetics, in which the kinetic orders of a reaction have positive integer values. However, recent theoretical and experimental advances have shown that the assumptions inherent in this classical framework are invalid under a variety of conditions in which the reaction environment may be considered nonideal. A good example is provided by reactions that are spatially constrained and diffusion limited. Bimolecular reactions confined within two-dimensional membranes, one-dimensional channels or fractal surfaces in general exhibit kinetic orders that are noninteger. An appropriate framework for the study of these nonideal phenomena is provided by the Power-Law formalism, which includes as special cases the Mass-Action formalism of chemical kinetics and the Michaelis-Menten formalism of enzyme kinetics. The Power-Law formalism is an appropriate representation not only for fractal kinetics per se, but also for other nonideal kinetic phenomena, provided the range of variation in concentration is not too large. After defining some elementary concepts of molecular recognition, and showing how these are manifested in classical kinetic terms, this paper contrasts the implications of classical and fractal kinetics in a few simple cases. The principal distinction lies in the ability of fractal kinetics to nonlinearly transform, rather than proportionally transmit, the input S/N ratio. As a consequence, fractal kinetics create a threshold for the input signal below which no recognition occurs and above which amplified recognition takes place.(ABSTRACT TRUNCATED AT 250 WORDS)
Enzymes↗
Subunit coupling and kinetic co-operativity of polymeric enzymes. Amplification, attenuation and inversion effects.
The principles of structural kinetics, as applied to dimeric enzymes, allow us to understand how the strength of subunit coupling controls both substrate-binding co-operativity, under equilibrium conditions, and kinetic co-operativity, under steady state conditions. When subunits are loosely coupled, positive substrate-binding co-operativity may result in either an inhibition by excess substrate or a positive kinetic co-operativity. Alternatively, negative substrate-binding co-operativity is of necessity accompanied by negative kinetic co-operativity. Whereas the extent of negative kinetic co-operativity is attenuated with respect to the corresponding substrate-binding co-operativity, the positive kinetic co-operativity is amplified with respect to that of the substrate-binding co-operativity. Strong kinetic co-operativity cannot be generated by a loose coupling of subunits. If subunit is propagated to the other, the dimeric enzyme may display apparently surprising co-operativity effects. If the strain of the active sites generated by subunit coupling is relieved in the non-liganded and fully-liganded states, both substrate-binding co-operativity and kinetic co-operativity cannot be negative. If the strain of the active sites however, is not relieved in these states, negative substrate-binding co-operativity is accompanied by either a positive or a negative co-operativity. The possible occurrence of a reversal of kinetic co-operativity, with respect to substrate-binding co-operativity, is the direct consequence of quaternary constraints in the dimeric enzyme. Moreover, tight coupling between subunits may generate a positive kinetic co-operativity which is not associated with any substrate-binding co-operativity. In other words a dimeric enzyme may well bind the substrate in a non co-operative fashion and display a positive kinetic co-operativity generated by the strain of the active sites.
Allosteric Regulation↗
The 3H-leucine tracer: its use in kinetic studies of plasma lipoproteins.
3H-leucine administered as a bolus has been widely used as a tracer in kinetic investigations of protein synthesis and secretion. After intravenous injection, plasma specific radioactivity decays over several orders of magnitude during the first half-day, followed by a slow decay lasting a number of weeks that results from recycling of the leucine tracer as proteins are degraded and 3H-leucine reenters the plasma pool. In studies in which kinetic data are analyzed by mathematical compartmental modeling, plasma leucine activity is generally used as a forcing function to drive the input of 3H-leucine into the protein synthesis pathway. 3H-leucine is an excellent tracer during the initial hours of rapidly decreasing plasma activity; thereafter, reincorporation of recycled tracer into new protein synthesis obscures the tracer data from proteins with slower turnover rates. Thus, for proteins such as plasma albumin and apolipoprotein (apo) A-I, this tracer is unsatisfactory for measuring fractional catabolic (FCR) and turnover rates. By contrast, the kinetics of plasma very-low-density lipoprotein (VLDL)-apoB, a protein with a residence time of approximately 5 hours, are readily measured, since kinetic parameters of this protein can be determined by the time plasma leucine recycling becomes established. However, measurement of VLDL-apoB specific radioactivity extending up to 2 weeks provides further data on the kinetic tail of VLDL-apoB. Were plasma leucine a direct precursor for the leucine in VLDL-apoB, the kinetics of the plasma tracer should determine the kinetics of the protein. However, this is not the case, and the deviations from linearity are interpreted in terms of (1) the dilution of plasma leucine in the liver by unlabeled dietary leucine; (2) the recycling of hepatocellular leucine from proteins within the liver, where recycled cellular leucine does not equilibrate with plasma leucine; and (3) a "hump" in the kinetic data of VLDL-apoB, which we interpret to reflect recycling or retention of a portion of the apoB protein within the hepatocyte, with its subsequent secretion. Because hepatocellular tRNA is the immediate precursor for synthesis of these secretory proteins, its kinetics should be used as the forcing function to drive the modeling of this system. The VLDL-apoB tail contains the information needed to modify the plasma leucine data, to provide an appropriate forcing function when using 3H-leucine as a tracer of apolipoprotein metabolism. This correction is essential when using 3H-leucine as a tracer for measuring low-density lipoprotein (LDL)-apoB kinetics. The 3H-leucine tracer also highlights the importance of recognizing the difference between plasma and system residence times, the latter including the time the tracer resides within exchanging extravascular pools. The inability to determine these fractional exchange coefficients for apoA-I and albumin explains the failure of this tracer in kinetic studies of these proteins. For apoB-containing lipoproteins, plasma residence times are generally determined, and these measurements can be made satisfactorily with 3H-leucine.
Apolipoprotein A-I↗
Pig heart lipoamide dehydrogenase: solvent equilibrium and kinetic isotope effects.
Lipoamide dehydrogenase is a flavoprotein which catalyzes the reversible oxidation of dihydrolipoamide, Lip(SH)2, by NAD+. The ping-pong kinetic mechanism involves stable oxidized and two-electron-reduced forms. We have investigated the rate-limiting nature of proton transfer steps in both the forward and reverse reactions catalyzed by the pig heart enzyme by using a combination of alternate substrates and solvent kinetic isotope effect studies. With NAD+ as the variable substrate, and at a fixed, saturating concentration of either Lip(SH)2 or DTT, inverse solvent kinetic isotope effects of 0.68 +/- 0.05 and 0.71 +/- 0.05, respectively, were observed on V/K. Solvent kinetic isotope effects on V of 0.91 +/- 0.07 and 0.69 +/- 0.02 were determined when Lip(SH)2 or DTT, respectively, was used as reductant. When Lip(SH)2 or DTT was used as the variable substrate, at a fixed concentration of NAD+, solvent kinetic isotope effects of 0.74 +/- 0.06 and 0.51 +/- 0.04, respectively, were observed on V/K for these substrates. Plots of the kinetic parameters versus mole fraction D2O (proton inventories) were linear in all cases. Solvent kinetic isotope effect measurements performed in the reverse direction using NADH as the variable substrate showed equivalent, normal solvent kinetic isotope effects on V/KNADH when oxidized lipoamide, lipoic acid, or DTT were present at fixed, saturating concentrations. Solvent kinetic isotope effects on V were equal to 1.5-2.1. When solvent kinetic isotope effect measurements were performed using the disulfide substrates lipoamide, lipoic acid, or DTT as the variable substrates, normal kinetic isotope effects on V/K of 1.3-1.7 were observed.(ABSTRACT TRUNCATED AT 250 WORDS)
Animals↗