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PubMed1996

Enzyme kinetic modelling as a tool to analyse the behaviour of cytochrome P450 catalysed reactions: application to amitriptyline N-demethylation.

1. To determine kinetic parameters (Vmax, K(m)) for cytochrome P450 (CYP) mediated metabolic pathways, nonlinear least squares regression is commonly used to fit a model equation (e.g., Michaelis Menten [MM]) to sets of data points (reaction velocity vs substrate concentration). This method can also be utilized to determine the parameters for more complex mechanisms involving allosteric or multi-enzyme systems. Akaike's Information Criterion (AIC), or an estimation of improvement of fit as successive parameters are introduced in the model (F-test), can be used to determine whether application of more complex models is helpful. To evaluate these approaches, we have examined the complex enzyme kinetics of amitriptyline (AMI) N-demethylation in vitro by human liver microsomes. 2. For a 15-point nortriptyline (NT) formation rate vs substrate (AMI) concentration curve, a two enzyme model, consisting of one enzyme with MM kinetics (Vmax = 1.2 nmol min-1 mg-1, K(m) = 24 microM) together with a sigmoidal component (described by an equation equivalent to the Hill equation for cooperative substrate binding; Vmax = 2.1 nmol min-1 mg-1, K' = 70 microM; Hill exponent n = 2.34), was favoured according to AIC and the F-test. 3. Data generated by incubating AMI under the same conditions but in the presence of 10 microM ketoconazole (KET), a CYP3A3/4 inhibitor, were consistent with a single enzyme model with substrate inhibition (Vmax = 0.74 nmol min-1 mg-1, K(m) = 186 microM, K1 = 0.0028 microM-1). 4. Sulphaphenazole (SPA), a CYP2C9 inhibitor, decreased the rate of NT formation in a concentration dependent manner, whereas a polyclonal rat liver CYP2C11 antibody, inhibitory for S-mephenytoin 4'-hydroxylation in humans, had no important effect on this reaction. 5. Incubation of AMI with 50 microM SPA resulted in a curve consistent with a two enzyme model, one with MM kinetics (Vmax = 0.72 nmol min-1 mg-1, K(m) = 54 microM) the other with 'Hill-kinetics' (Vmax = 2.1 nmol min-1 mg-1, K' = 195 microM; n = 2.38). 6. A fourth data-set was generated by incubating AMI with 10 microM KET and 50 microM SPA. The proposed model of best fit describes two activities, one obeying MM-kinetics (Vmax = 0.048 nmol min-1 mg-1, K(m) = 7 microM) and the other obeying MM kinetics but with substrate inhibition (Vmax = 0.8 nmol min-1 mg-1, K(m) = 443 microM, K1 = 0.0041 microM-1). 7. The combination of kinetic modelling tools and biological data has permitted the discrimination of at least three CYP enzymes involved in AMI N-demethylation. Two are identified as CYP3A3/4 and CYP2C9, although further work in several more livers is required to confirm the participation of the latter.

Amitriptyline↗

PubMed2003

Cerebral and lung kinetics of morphine in conscious sheep after short intravenous infusions.

BACKGROUND: The analgesic effects of morphine are delayed relative to its concentration in blood. The rate of equilibration of morphine between blood and brain may contribute to this delay, but the kinetics of this process have not been modelled. This was determined in conscious instrumented sheep. The lung kinetics of morphine were also determined given their importance in defining systemic kinetics after i.v. bolus administration. METHODS: Sheep were given short i.v. infusions of morphine (30 mg over 4 min). Cerebral kinetics were inferred from arterio-sagittal sinus concentration gradients and cerebral blood flow, and lung kinetics from the pulmonary artery-aortic gradient and cardiac output. These data were fitted to flow- and membrane-limited models of the kinetics in each organ. RESULTS: Morphine had minimal cardiovascular effects, did not alter cerebral blood flow and caused insignificant respiratory depression. Lung kinetics were best described by a single distribution volume (2036 ml) with a first-order loss (1370 ml min(-1)), which was attributed to deep distribution. The cerebral kinetics of morphine were characterized by a significant permeability barrier. Permeability across the barrier (7.44 ml min(-1)) was estimated with good precision, and was approximately one-fifth of the nominal cerebral blood flow. The distribution volume of morphine in the brain was estimated with less precision, but was described by a brain:blood partition coefficient of approximately 1.4. The time required for 50% equilibration between brain and blood concentrations was approximately 10.3 min. CONCLUSION: The cerebral equilibration of morphine was relatively slow, and was characterized by significant membrane limitation.

Analgesics, Opioid↗

PubMed2004

Comparison of oxygen uptake kinetics during knee extension and cycle exercise.

The knee extension exercise (KE) model engenders different muscle and fiber recruitment patterns, blood flow, and energetic responses compared with conventional cycle ergometry (CE). This investigation had two aims: 1) to test the hypothesis that upright two-leg KE and CE in the same subjects would yield fundamentally different pulmonary O(2) uptake (pVo(2)) kinetics and 2) to characterize the muscle blood flow, muscle Vo(2) (mVo(2)), and pVo(2) kinetics during KE to investigate the rate-limiting factor(s) of pVo(2) on kinetics and muscle energetics and their mechanistic bases after the onset of heavy exercise. Six subjects performed KE and CE transitions from unloaded to moderate [< ventilatory threshold (VT)] and heavy (>VT) exercise. In addition to pVo(2) during CE and KE, simultaneous pulsed and echo Doppler methods, combined with blood sampling from the femoral vein, were used to quantify the precise temporal profiles of femoral artery blood flow (LBF) and mVo(2) at the onset of KE. First, the gain (amplitude/work rate) of the primary component of pVo(2) for both moderate and heavy exercise was higher during KE ( approximately 12 ml.W(-1).min(-1)) compared with CE ( approximately 10), but the time constants for the primary component did not differ. Furthermore, the mean response time (MRT) and the contribution of the slow component to the overall response for heavy KE were significantly greater than for CE. Second, the time constant for the primary component of mVo(2) during heavy KE [25.8 +/- 9.0 s (SD)] was not significantly different from that of the phase II pVo(2). Moreover, the slow component of pVo(2) evident for the heavy KE reflected the gradual increase in mVo(2). The initial LBF kinetics after onset of KE were significantly faster than the phase II pVo(2) kinetics (moderate: time constant LBF = 8.0 +/- 3.5 s, pVo(2) = 32.7 +/- 5.6 s, P < 0.05; heavy: LBF = 9.7 +/- 2.0 s, pVo(2) = 29.9 +/- 7.9 s, P < 0.05). The MRT of LBF was also significantly faster than that of pVo(2). These data demonstrate that the energetics (as gain) for KE are greater than for CE, but the kinetics of adjustment (as time constant for the primary component) are similar. Furthermore, the kinetics of muscle blood flow during KE are faster than those of pVo(2), consistent with an intramuscular limitation to Vo(2) kinetics, i.e., a microvascular O(2) delivery-to-O(2) requirement mismatch or oxidative enzyme inertia.

Adolescent↗

PubMedDate not supplied

Patellofemoral stresses during open and closed kinetic chain exercises. An analysis using computer simulation.

Rehabilitation of the symptomatic patellofemoral joint aims to strengthen the quadriceps muscles while limiting stresses on the articular cartilage. Some investigators have advocated closed kinetic chain exercises, such as squats, because open kinetic chain exercises, such as leg extensions, have been suspected of placing supraphysiologic stresses on patellofemoral cartilage. We performed computer simulations on geometric data from five cadaveric knees to compare three types of open kinetic chain leg extension exercises (no external load on the ankle, 25-N ankle load, and 100-N ankle load) with closed kinetic chain knee-bend exercises in the range of 20 degrees to 90 degrees of flexion. The exercises were compared in terms of the quadriceps muscle forces, patellofemoral joint contact forces and stresses, and "benefit indices" (the ratio of the quadriceps muscle force to the contact stress). The study revealed that, throughout the entire flexion range, the open kinetic chain stresses were not supraphysiologic nor significantly higher than the closed kinetic chain exercise stresses. These findings are important for patients who have undergone an operation and may feel too unstable on their feet to do closed chain kinetic chain exercises. Open kinetic chain exercises at low flexion angles are also recommended for patients whose proximal patellar lesions preclude loading the patellofemoral joint in deeper flexion.

Computer Simulation↗

PubMedDate not supplied

Open versus closed chain kinetic exercises after anterior cruciate ligament reconstruction. A prospective randomized study.

We conducted a prospective, randomized study of open and closed kinetic chain exercises during accelerated rehabilitation after anterior cruciate ligament reconstruction to determine if closed kinetic chain exercises are safe and if they offer any advantages over conventional rehabilitation. The closed kinetic chain group used a length of elastic tubing, the Sport Cord, to perform weightbearing exercises and the open kinetic chain group used conventional physical therapy equipment. Results are reported with a minimum 1-year followup (mean, 19 months). Pre- and postoperative evaluation included the Lysholm knee function scoring scale, Tegner activity rating scale and KT-1000 arthrometer measurements. Overall, stability was restored in over 90% of the knees. Preoperative patellofemoral pain was reduced significantly; 95% of the patients had a full range of motion. The closed kinetic chain group had lower mean KT-1000 arthrometer side-to-side differences, less patellofemoral pain, was generally more satisfied with the end result, and more often thought they returned to normal daily activities and sports sooner than expected. We concluded that closed kinetic chain exercises are safe and effective and offer some important advantages over open kinetic chain exercises. As a result of this study, we now use the closed kinetic chain protocol exclusively after anterior cruciate ligament reconstruction.

Adolescent↗

PubMedDate not supplied

A comparison of tibiofemoral joint forces and electromyographic activity during open and closed kinetic chain exercises.

We chose to investigate tibiofemoral joint kinetics (compressive force, anteroposterior shear force, and extension torque) and electromyographic activity of the quadriceps, hamstring, and gastrocnemius muscles during open kinetic chain knee extension and closed kinetic chain leg press and squat. Ten uninjured male subjects performed 4 isotonic repetitions with a 12 repetition maximal weight for each exercise. Tibiofemoral forces were calculated using electromyographic, kinematic, and kinetic data. During the squat, the maximal compressive force was 6139 +/- 1708 N, occurring at 91 degrees of knee flexion; whereas the maximal compressive force for the knee extension exercise was 4598 +/- 2546 N (at 90 degrees knee flexion). During the closed kinetic chain exercises, a posterior shear force (posterior cruciate ligament stress) occurred throughout the range of motion, with the peak occurring from 85 degrees to 105 degrees of knee flexion. An anterior shear force (anterior cruciate ligament stress) was noted during open kinetic chain knee extension from 40 degrees to full extension; a peak force of 248 +/- 259 N was noted at 14 degrees of knee flexion. Electromyographic data indicated greater hamstring and quadriceps muscle co-contraction during the squat compared with the other two exercises. During the leg press, the quadriceps muscle electromyographic activity was approximately 39% to 52% of maximal velocity isometric contraction; whereas hamstring muscle activity was minimal (12% maximal velocity isometric contraction). This study demonstrated significant differences in tibiofemoral forces and muscle activity between the two closed kinetic chain exercises, and between the open and closed kinetic chain exercises.

Adult↗

PubMed2005

Evidence for complexity at the nanometer scale of radiation-induced DNA DSBs as a determinant of rejoining kinetics.

The rejoining kinetics of double-stranded DNA fragments, along with measurements of residual damage after postirradiation incubation, are often used as indicators of the biological relevance of the damage induced by ionizing radiation of different qualities. Although it is widely accepted that high-LET radiation-induced double-strand breaks (DSBs) tend to rejoin with kinetics slower than low-LET radiation-induced DSBs, possibly due to the complexity of the DSB itself, the nature of a slowly rejoining DSB-containing DNA lesion remains unknown. Using an approach that combines pulsed-field gel electrophoresis (PFGE) of fragmented DNA from human skin fibroblasts and a recently developed Monte Carlo simulation of radiation-induced DNA breakage and rejoining kinetics, we have tested the role of DSB-containing DNA lesions in the 8-kbp-5.7-Mbp fragment size range in determining the DSB rejoining kinetics. It is found that with low-LET X rays or high-LET alpha particles, DSB rejoining kinetics data obtained with PFGE can be computer-simulated assuming that DSB rejoining kinetics does not depend on spacing of breaks along the chromosomes. After analysis of DNA fragmentation profiles, the rejoining kinetics of X-ray-induced DSBs could be fitted by two components: a fast component with a half-life of 0.9+/-0.5 h and a slow component with a half-life of 16+/-9 h. For alpha particles, a fast component with a half-life of 0.7+/-0.4 h and a slow component with a half-life of 12+/-5 h along with a residual fraction of unrepaired breaks accounting for 8% of the initial damage were observed. In summary, it is shown that genomic proximity of breaks along a chromosome does not determine the rejoining kinetics, so the slowly rejoining breaks induced with higher frequencies after exposure to high-LET radiation (0.37+/-0.12) relative to low-LET radiation (0.22+/-0.07) can be explained on the basis of lesion complexity at the nanometer scale, known as locally multiply damaged sites.

Cells, Cultured↗

PubMed2003

Atypical enzyme kinetics: their effect on in vitro-in vivo pharmacokinetic predictions and drug interactions.

The most common method for estimating a drug's in vivo clearance from in vitro data has involved using the classical Michaelis-Menten model to describe the observed in vitro data and from this estimate intrinsic clearance. This process assumes that the drug obeys standard enzyme kinetics that can be described by this model. However, the observation of atypical enzyme kinetics, particularly involving cytochrome P450 enzymes, has become relatively common, and occurrences have been reported with virtually all of the cytochrome P450 isoforms. Since predictions of a drug's in vivo clearance are made based on kinetic parameters observed during in vitro experiments (in vitro estimated intrinsic clearance), mis-identification of the drug's kinetic profile can lead to inaccurate predictions of intrinsic clearance. In addition to these potential ramifications of in vitro-in vivo predictions, information is becoming available concerning the in vivo implications of drugs that exhibit atypical kinetic profiles. This review will discuss the various types of atypical kinetic profiles that may be observed during in vitro experiments, hypotheses for why they may occur, examples of how a drug's intrinsic clearance may be mis-estimated when atypical kinetics are incorrectly assessed, and the potential ramifications of these issues for prediction of drug interactions. Furthermore, potential, artifactual causes of atypical kinetic phenomena will be described.

Algorithms↗

PubMed1999

Kinetic and static fixation methods in automated threshold perimetry.

PURPOSE: Static fixation is the standard method for stabilizing the eye during automated perimetry. Kinetic fixation is an alternative for fixation control in which the eye follows a moving target. This study was conducted to evaluate the fixation accuracy of static and kinetic fixation perimetry and to determine their ability to detect the absolute scotoma of the physiologic blind spot. METHODS: The 71 patients with early glaucomatous field loss (mean age 65 years) and 45 control subjects (mean age 57 years) recruited from five clinical sites underwent threshold testing on the Dicon perimeter (kinetic fixation; Vismed, San Diego, CA) and Humphrey Field Analyzer (static fixation). The frequency of Heijl-Krakau fixation catch-trial errors was used as an indicator of fixation accuracy, and the measured sensitivity at the physiologic blind spot was used as an indicator of perimetric accuracy. RESULTS: In patients with glaucoma, the frequency of fixation errors was significantly greater for kinetic fixation (17.2%) than for static fixation (10.2%). In the control group, the frequency of fixation errors also was significantly greater for kinetic fixation (27.5%) than for static fixation (12.6%). The threshold at the presumed location of the blind spot (15 degrees temporal, 3 degrees inferior from fixation) was 14.8 dB using kinetic fixation versus 4.0 dB with static fixation in patients with glaucoma, and 18.5 dB using kinetic fixation versus 2.5 dB using static fixation in the control group. CONCLUSION: Relative to static fixation, kinetic fixation was associated with fixation inaccuracy and underestimation of the absolute scotoma at the physiologic blind spot.

Adult↗

PubMed2000

A kinetic chain approach for shoulder rehabilitation.

OBJECTIVE: To introduce an approach to shoulder rehabilitation that integrates the kinetic chain throughout the rehabilitation program while providing the theoretical rationale for this program. BACKGROUND: The focus of a typical rehabilitation program is to identify and treat the involved structures. However, in activities of sport and daily life, the body does not operate in isolated segments but rather works as a dynamic unit. Recently, rehabilitation programs have emphasized closed kinetic chain exercises, core-stabilization exercises, and functional programs. These components are implemented as distinct entities and are used toward the end of the rehabilitation program. DESCRIPTION: Kinetic chain shoulder rehabilitation incorporates the kinetic link biomechanical model and proximal-to-distal motor-activation patterns with proprioceptive neuromuscular facilitation and closed kinetic chain exercise techniques. This approach focuses on movement patterns rather than isolated muscle exercises. Patterns sequentially use the leg, trunk, and scapular musculature to activate weakened shoulder musculature, gain active range of motion, and increase strength. The paradigm of kinetic chain shoulder rehabilitation suggests that functional movement patterns and closed kinetic chain exercises should be incorporated throughout the rehabilitation process. CLINICAL ADVANTAGES: The exercises in this approach are consistent with biomechanical models, apply biomechanical and motor control theory, and work toward sport specificity. The exercises are designed to stimulate weakened tissue by motion and force production in the adjacent kinetic link segments.

Journal Article↗

PubMed1998

Enzyme kinetic reactions and fluorochrome uptake rates measured in individual cells by laser scanning cytometry.

This study was designed to explore the utility of a microscope-based cytofluorometer, the laser scanning cytometer (LSC), for time-resolved kinetic measurements. This instrument measures fluorescence of individual cells rapidly, with high sensitivity and accuracy. Also recorded in a list mode fashion are the spatial X-Y coordinates of the cell on the slide as well as the time of individual cell measurement. Repeated measurement of each of a group of cells within a selected area of the slide, thus, yields information on their fluorescence parameters (integrated value, maximal pixel intensity, or fluorescence area) as a function of time. Using the fluorogenic substrate di-(leucyl)-rhodamine 110, we measured the kinetic activity of L-aminopeptidase in HL-60 cells and in monocytes, granulocytes, and lymphocytes from human blood. Likewise, the rate of fluorescein diacetate (FDA) hydrolysis by esterases was measured in HL-60 cells. Also studied was the rate of uptake of the lysosomo-trophic fluorochrome acridine orange (AO) by human leukocytes. Several hundred cells per sample were measured rapidly with a time resolution of 20-60 s. The resolution was inversely proportional to the number of cells within the measured population. The kinetic curves constructed for individual cells had clearly defined slope and plateau regions. During data analysis the kinetic plots were matched with the respective cells; the latter were identified by their position on the slide and classified by their fluorescence or image after staining with Giemsa. Great intercellular variability was noted in enzyme kinetics among cells of the same type, and differences were seen in rate of AO uptake between granulocytes, monocytes, and lymphocytes. Fluorescence fading and recovery was observed especially in the case of FDA hydrolysis and AO uptake. Our results indicate that LSC can be used to rapidly measure the rate of uptake or accumulation of a particular fluorochrome or the kinetics of enzymatic reactions in individual cells of large populations to reveal intercellular variability or the presence of a cell subpopulation with different kinetic properties.

Acridine Orange↗

PubMed2001

Kinetic aspects of the Maillard reaction: a critical review.

The literature concerning the kinetics of the Maillard reaction was critically discussed according to the initial, intermediate and advanced stages, as this is the way the Maillard reaction is traditionally analysed. For each stage, a division is made between simple kinetics and complex kinetics. Simple kinetics means that the general rate law is used and results are reported as zero-, first- or second-order reactions (sometimes a fractional order). It is emphasized that this approach for a complex reaction as the Maillard reaction only results in a mathematical fit procedure, not in mechanistic insight. The rate constants and activation energies derived are in fact composed of several elementary rate constants. With complex kinetics, i.e. trying to establish the kinetics for individual reaction steps, more mechanistic information can be extracted. However, there are conflicting results in literature and the interpretation is not always correct, as is shown in several examples. A summary of activation energies reported for the various stages in the Maillard reaction reveals large discrepancies, probably reflecting strong effects of experimental conditions on results that are obtained. Careful control of experimental conditions and proper kinetic analysis of the various stages in the Maillard reaction should lead to more consistent results in the future.

Carbohydrate Metabolism↗

PubMed1982

Kinetic alterations of the red cell membrane phosphatase in alpha- and beta-thalassemia.

We studied the red cell membrane neutral phosphatase, which is part of the Na+K+ ATPase, in several types of oxidative hemolytic anemias. We used an artificial substrate, the p-nitrophenylphosphate. In controls and in patients heterozygous for various unstable hemoglobins (Hb Hope, Hb Köln, or Hb Hammersmith), the kinetics were of the Michaelis-Menten type. On the contrary, in nearly all patients with alpha- or beta-thalassemia, the kinetics displayed an abnormally biphasic character. The apparent Michaelis constant (KMapp) was significantly decreased. The biphasic character correlated with the imbalance of globin chain synthesis. The beta-mercaptoethanol markedly increased Vmax in controls, but had little effect on the biphasic kinetics. Omission of K+ abolished the biphasic kinetics. The abnormal kinetics failed to appear with another artificial substrate, the 4-methylumbelliferylphosphate, nor did it appear with ATP, the natural substrate. In vitro, H2O2 treatment of normal and thalassemic red cells was unable to induce or exaggerate, respectively, the biphasic kinetics, but generated alterations of a different nature. We suggest that the various kinetic alterations of the phosphatase in thalassemic syndromes originate from the imbalance of globin chain synthesis. However, the involvement of an oxidative process remains to be demonstrated.

Erythrocyte Membrane↗

PubMed2005

Biosynthesis reaction mechanism and kinetics of deoxynucleoside triphosphates, dATP and dGTP.

The enzyme reaction mechanism and kinetics for biosyntheses of deoxyadenosine triphosphate (dATP) and deoxyguanosine triphosphate (dGTP) from the corresponding deoxyadenosine diphosphate (dADP) and deoxyguanosine diphosphate (dGDP) catalyzed by pyruvate kinase were studied. A kinetic model for this synthetic reaction was developed based on a Bi-Bi random rapid equilibrium mechanism. Kinetic constants involved in this pyruvate kinase catalyzed phosphorylation reactions of deoxynucleoside diphosphates including the maximum reaction velocity, Michaelis-Menten constants, and inhibition constants for dATP and dGTP biosyntheses were experimentally determined. These kinetic constants for dATP and dGTP biosyntheses are of the same order of magnitude but significantly different between the two reactions. Kinetic constants involved in ATP and GTP biosyntheses as reported in literature are about one order of magnitude different from those involved in dATP and dGTP biosyntheses. This enzyme reaction requires Mg2+ ion and the optimal Mg2+ concentration was also determined. The experimental results showed a very good agreement with the simulation results obtained from the kinetic model developed. This kinetic model can be applied to the practical application of a pyruvate kinase reaction system for production of dATP and dGTP. There is a significant advantage of using enzymatic biosyntheses of dATP and dGTP as compared to the chemical method that has been in commercial use.

Biotechnology↗

PubMedDate not supplied

The kinetics of baboon fibrinogen adsorption to polymers: in vitro and in vivo studies.

Fibrinogen adsorption on polymers from blood may mediate or potentiate thrombosis because of its involvement in both the intrinsic clotting system and the formation of platelet aggregates. While the kinetics of fibrinogen adsorption from plasma in vitro have previously been found to be very different on polar and nonpolar surfaces [T. A Horbett, "The kinetics of adsorption of plasma proteins to a series of hydrophilic-hydrophobic copolymers," ACS Org. Coat. Plas. Chem. 40, 642-646 (1979)] the significance of this difference with respect to thrombogenesis in vivo has not been clarified. In this study, the kinetics of deposition of baboon 125I fibrinogen from plasma in vitro or from blood in vivo on a series of polymers was measured. The polymers chosen for this study had previously been found to have a large range in surface polarity and reactivity in the in vivo baboon shunt model. The kinetics of fibrinogen adsorption in vitro were observed to be of three types, depending on the polymer: high initial adsorption decreasing to a lower steady state value; constant throughout the time course; low initial adsorption rising steadily to a plateau value. In vivo, fibrinogen deposition kinetics were of two types: low, constant deposition throughout the time course, independent of heparinization; low deposition initially followed by a second phase of greatly increased deposition (probably as fibrin) which was prevented or greatly decreased by heparinizing the animals. Polymers for which fibrinogen adsorption increased to a plateau in vitro were found to have a heparin inhibitable second phase of enhanced in vivo fibrinogen deposition. These polymers also have been found in previous studies to enhance the rate of platelet destruction when used as in vivo shunts on baboons. Conversely, most polymers with high initial in vitro fibrinogen adsorption followed by a decrease had low fibrinogen deposition behavior in vivo and were also minimally destructive of platelets. The adsorption kinetics of fibrinogen to polymers from blood in vivo and in vitro and the consumption of platelets in vivo induced by the polymers all vary with polymer polarity. More polar polymers had in vitro fibrinogen kinetics characterized by a rise to a plateau, in vivo fibrinogen deposition characterized by a second stage of great increase inhibitable by heparin, and enhanced platelet consumption. The correlation of three separate indicators of surface thrombogenicity with surface polarity suggests that more polar materials may be more thrombogenic because of an influence on the way in which fibrinogen interacts with these surfaces.

Adsorption↗

PubMedDate not supplied

Kinetic determinations of molecular interactions using Biacore--minimum data requirements for efficient experimental design.

Reliable kinetic estimates can be obtained from significantly less data than is commonly used today, particularly in the characterization of 1:1 interactions involving low molecular weight compounds and proteins. We have designed a rational and cost-effective strategy to determine kinetic constants using Biacore's surface plasmon resonance-based biosensors and show that the number of measurements necessary for accurate kinetic determinations can be greatly reduced, increasing sample throughput and saving sample material. Simulated and measured data for a range of possible 1:1 interactants were studied to find the minimum requirements of a data set for kinetic analysis. The results showed that kinetic constants in the region 10(4) < k(a) < 10(7) M(-1) s(-1) (association) and 10(-4) < k(d) < 10(-1) s(-1) (dissociation) could easily be determined in a 1:1 interaction model. Owing to the information-dense nature of Biacore data, only two sample concentrations were necessary to reliably determine the kinetics. A standard sample concentration series consisting of 10-fold dilutions between approximately 10 microM and approximately 1 nM consistently provided at least two concentrations with sufficient information about the interaction in this region. Determinations of the constants became increasingly unreliable outside this region. If the rate constants prove to be outside the specified region or the data fits poorly to the 1:1-MTL model, more experiments are required. General recommendations for the design of a cost-effective assay to deliver reliable kinetic measurements are provided.

Biosensing Techniques↗

PubMed1993

Synthetic chimeras of mouse growth factor-associated glandular kallikreins. I. Kinetic properties.

A series of six chimeric proteins, composed of fragments corresponding to either one or the other of the growth factor-associated mouse glandular kallikreins-epidermal growth factor binding protein (EGF-BP) and the gamma-subunit of nerve growth factor (gamma-NGF)--were expressed in Escherichia coli and isolated, and their kinetic properties were characterized. The assembly of these synthetic proteases involved the substitution of regions of the proteins containing four specific surface loops that have been postulated to influence both kinetic specificity and the formation of growth factor complexes. The substrates utilized in the kinetic characterization of these chimeric kallikreins were tripeptide nitroanilides representing carboxyl termini of both the EGF and beta-NGF mature hormones, putative processing sites for these kallikreins in the precursors. Characterization of these hybrid enzymes demonstrates that Km and kcat kinetic constants may be independently affected by the regions utilized in construction of these chimeric kallikreins. Specifically, loop 1, located in the amino terminal region (Bode, W., et al., J. Mol. Biol. 164, 237-282, 1983), in gamma-NGF enhanced the kcat for substrates containing threonine in the P2 position, as is the case during the processing of the carboxy terminus of the beta-NGF precursor. Also, the central regions of the kallikreins containing loop 2 and the kallikrein loop dictated the generally inverted Km and kcat kinetic constants observed between EGF-BP and gamma-NGF. Finally, in gamma-NGF the autolysis loop, found in the carboxyl terminal region, functions to lower the Km kinetic constant for a variety of substrates. The results allow previously characterized kinetic differences between EGF-BP and gamma-NGF to be interpreted in terms of specific regions of the proteins and identify a subset of amino acid positions responsible for these functional characteristics.

Amino Acid Sequence↗

PubMed1997

Studies of protein interactions by biosensor technology: an alternative approach to the analysis of sensorgrams deviating from pseudo-first-order kinetic behavior.

A procedure for evaluating the thermodynamic equilibrium constant by kinetic analysis of sensorgrams which deviate from the pseudo-first-order kinetic behavior predicted for 1:1 interactions between ligate and affinity sites on the sensor surface is described. This analysis employs quantitative expressions that are used in conventional kinetic characterization of protein interactions by biosensor technology, but with the equilibrium sensorgram response fixed at a predetermined magnitude. Simulated sensorgrams for situations in which the aberrant kinetic behavior reflects (i) heterogeneity of affinity sites and (ii) isomerization of the complex between ligate and affinity sites are used to explore the feasibility of the approach. Its application is then illustrated with BIAcore studies of the interaction between the Fab fragment of an anti-paraquat monoclonal antibody and immobilized antigen in the form of a paraquat analog attached covalently to the sensor surface. Studies with an extremely high degree of antigen substitution on the sensor surface yielded sensorgrams that deviated markedly from pseudo-first-order kinetic behavior. However, they yielded the same binding constant (3 x 10(6) M-1) as the value deduced by conventional analysis of sensorgrams that conformed with pseudo-first-order kinetics because of a much lower concentration of immobilized antigen on the sensor surface. Such identity of binding constants eliminates heterogeneity of immobilized paraquat sites as the likely source of the aberrant kinetic behavior.

Antibodies, Monoclonal↗