Search PubMed⌕ Search

SEARCH · Search PubMed

Results for “Enzyme kinetics”

Search indexed PubMed citations on genomics, clinical trials, systematic reviews and public health. Explore titles, authors and supplied subject terms, then open the PubMed record.

Quote a phrase for an exact phrase match. Source license links do not imply unrestricted reuse.

At least 163 records · Page 9Linked to original sources
PubMed1984

An inexpensive computerized enzyme kinetics system based on a Gilford spectrophotometer and an Apple IIe microcomputer.

A microcomputer-controlled data acquisition system for spectrophotometric enzyme kinetics measurements has been assembled. The system uses an Apple IIe computer which is interfaced to the binary coded decimal output of a Gilford spectrophotometer. No analog-to-digital converter had to be purchased. A BASIC program which collects timed absorbance readings every 500 ms, plots the data in real time, performs a linear regression of the data to measure the reaction rate, and calculates the enzyme activity concentration is given in full. Details describing the interfacing of the computer to the spectrophotometer are presented which will permit other laboratories to readily assemble their own systems using this hardware. Kinetic data acquired by the system are highly reproducible and agree well with data processed much more slowly by manual techniques from strip chart recordings.

Autoanalysis↗
PubMed1986

Enzyme kinetic studies from progress curves.

A method is described for fitting the velocities obtained from progress curves to a steady-state rate equation. It is based on the method of Markus & Plesser [(1981) in Kinetic Data Analysis: Design and Analysis of Enzyme and Kinetic Data (Edrenyi, ed.), pp. 317-339, Plenum Press, New York]. The obstacle of needing good initial estimates of kinetic parameters is removed by using the parameters provided graphically by a minor modification of the method of Yun & Suelter [(1977) Biochim, Biophys. Acta 480, 1-13]. This progress-curved-based method allows the same discrimination among rival models as do the initial-velocity-based methods, with a great saving of experimental time. The BASIC and FORTRAN 77 programs are deposited as Supplementary Publication SUP 50132 (17 pages) at the British Library (Lending Division), Boston Spa, Wetherby, West Yorkshire LS23 7BQ, U.K., from whom copies can be obtained on the terms indicated in Biochem. J. (1986) 233, 5-6.

Adenosine Deaminase↗
PubMed1998

Genotype-phenotype correlations in mucopolysaccharidosis type I using enzyme kinetics, immunoquantification and in vitro turnover studies.

Fibroblasts from 16 patients with known alpha-L-iduronidase gene mutations and different clinical phenotypes of mucopolysaccharidosis type I (MPS I) were investigated in order to establish genotype/phenotype correlations. Enzyme kinetic studies were performed using the specific alpha-L-iduronidase substrate iduronosyl anhydro[1-3H]mannitol-6-sulfate. Specific residual enzyme activities were estimated using the kinetic parameters and an immunoquantification assay which determines levels of alpha-L-iduronidase protein. Cells were cultured in the presence of [35S]sulfate and the in vivo degradation of accumulated labelled glycosaminoglycans measured after different chase times. Residual enzyme activity and different amounts of residual enzyme protein were present in extracts from 9 of 16 cell lines covering a wide spectrum of clinical severity. Catalytic capacity, calculated as the product of kcat/Km and ng iduronidase protein per mg cell protein, was shown in most cases to be directly related to the severity of clinical phenotype, with up to 7% of normal values for patients with the attenuated form of MPS I (Scheie) and less than 0.13% for severely affected patients (Hurler) In vitro turnover studies allowed further refinement of correlations between genotype and phenotype. Scheie disease compared to Hurler disease patients were shown to accumulate smaller amounts of glycosaminoglycans that were also turned over faster. A combination of turnover and residual enzyme data established a correlation between the genotype, the biochemical phenotype and the clinical course of this lysosomal storage disorder.

Cell Line↗
PubMed1986

Estimating enzyme kinetic parameters: a computer program for linear regression and non-parametric analysis.

An IBM computer program, WILMAN4, is described which calculates the estimates, Km, V and Km/V from initial velocity measurements according to one of four statistical methods. Three of these methods involve linear regression analysis using weights given by assuming: (i) constant absolute error (G.N. Wilkinson, 1961, Biochem J., 80, 324-332), (ii) constant relative error (G. Johansen and R. Lumry, 1961, C.R. Trav. Lab. Carlsberg, 32, 185-214) and (iii) an error function in between the above two cases. (A. Cornish-Bowden, 1976, Principles of Enzyme Kinetics, Butterworths Inc, Boston, Mass., pp. 168-193). The fourth method is a non-parametric procedure derived by Eisenthal and Cornish-Bowden (Biochim. Biophys. Acta, 532 (1974) 268-272). Residuals are obtained by subtracting the experimental and the calculated velocities. Outliers, or residuals which are greater than two experimental standard deviations, can be identified and removed from the data set. If the sequence of positive and negative signs of the residuals is random as determined by a statistical probability calculation, the data set is assumed to obey the Michaelis-Menten equation.

Biometry↗
PubMed1996

A cellular automata model of enzyme kinetics.

We have developed a cellular automata model of an enzyme reaction with a substrate in water. The model produces Michaelis-Menten kinetics with good Lineweaver-Burk plots. The variation in affinity parameters predicts that, in general, hydrophobic substrates are more reactive with enzymes, this attribute being more important than the relationship between enzyme and substrate. The ease of generation and the illustrative value of the model lead us to believe that cellular automata models have a useful role in the study of dynamic phenomena such as enzyme kinetics.

Automation↗
PubMed1991

Enzyme kinetic and inhibition analyses of cytochrome P450XVII, a protein with a bifunctional catalytic site. Quantification of effective substrate concentrations at the active site and their significance for intrinsic control of the hydroxylase/lyase reaction sequence.

An enzyme kinetic analysis of the cytochrome P450XVII-dependent testicular steroid-17 alpha-hydroxylase/17,20-lyase system was performed which catalyzes simultaneously, at one active site, the formation of androstenedione from progesterone (productive pathway, coordinated performance of both hydroxylase and lyase activities) and the formation of 17 alpha-hydroxyprogesterone as the intermediate (abortive pathway, isolated performance of hydroxylase activity only). Progesterone was used as the substrate and promegestone (17 alpha, 21-dimethyl-4,9(10)-pregnadiene-3,20-dione) or estradiol as competitive inhibitors, and description of the data was based on the discrimination of those alternative routes of progesterone metabolism. The overall catalytic activity of P450XVII obeyed Michaelis-Menten kinetics (control incubations: V = 440 nM/min with 80 nM P450XVII, Km = 140 nM), and KI values amounted to 6.8 microM for promegestone and to 23.5 microM for estradiol. In contrast, analysis of the rates of both abortive and productive events yielded curvilinear Eadie-Hofstee plots. The former presented an apparent negative cooperative behavior with nh = 0.78 in the region of Kh, while the latter presented an apparent substrate inhibition behavior with the maximal rate occurring at that progesterone concentration where nh for the abortive path reached its minimum. Consequently, the ratio of productive versus abortive catalytic events decreased with increasing substrate supply, but did not reach zero. Effective substrate concentrations [S]eff with respect to either the productive or the abortive pathway were derived from Hill plots. Linearization of Eadie-Hofstee plots and parallel calculation methods using these [S]eff instead of [S] yielded V = 236 nM/min and [S]eff(0.5) = 131 nM for the abortive events and V = 217 nM/min and [S]eff(0.5) = 38 nM for the productive events. Both promegestone and estradiol were identified as competitive inhibitors of either reaction after consideration of effective progesterone concentrations. With the inhibitors, ratios of productive versus abortive events were constantly higher at a given substrate concentration than in control incubations, although neither promegestone nor estradiol affected progesterone or 17 alpha-hydroxyprogesterone accumulation in the endoplasmic reticulum membrane compartment. Rather, a linear correlation between ratios of productive versus abortive events and the overall catalytic rate as a measure of E.S complex concentration was obtained irrespective of the absence or presence of inhibitor. It was therefore concluded that promegestone and estradiol inhibit access or binding of progesterone to P450XVII, whereas the relative efficiency of androgen formation is solely dictated by the local substrate concentration being effective with respect to E.S formation. A model of P450XVII function is presented which proposes that excess substrate at the active site of the P450XVII enzyme protein hinders a certain fraction of a putative transient intermediate from being retained at the bifunctional catalytic site with the consequence that it cannot be further processed to androgen.

Animals↗
PubMed1983

Fitting enzyme-kinetic data to V/K.

Kinetic data from enzyme-catalyzed reactions have been analyzed traditionally in terms of the Michaelis-Menten equation, which assumes that the maximal velocity (V) and the Michaelis constant (K) are the primary kinetic constants. But what is needed from most kinetic studies today is V/K. A new form of the equation is proposed which assumes that V and V/K are the primary kinetic constants: v = (V . S . V/K)/(V + S . V/K). Computer fittings of both experimental and simulated velocity data to both equations give results favoring the new equation.

Computers↗
PubMed1969

Observations on programs to estimate the parameters of enzyme kinetics.

This paper gives a critical account of the two major programs so far published specifically to estimate the parameters of enzyme kinetics. The dangers of submitting data to the programs without proper checks are discussed, and a screening test is described to identifty sets of data which may not be best fitted by a rectangular hyperbola.

Journal Article↗
PubMed1997

Molecular basis for deficient acetaminophen glucuronidation in cats. An interspecies comparison of enzyme kinetics in liver microsomes.

Cats are highly susceptible to acetaminophen toxicity because of deficient glucuronidation of this drug in vivo. The enzyme kinetic basis for this defect is unknown. Therefore, the kinetic properties of acetaminophen UDP-glucuronosyltransferase (acetaminophen-UGT) were investigated, using hepatic microsomes from cats (N = 4) compared with those of species that are less sensitive to acetaminophen intoxication including dogs (N = 4), humans (N = 4), and six other mammalian species (one liver from each). Gunn rats were also studied, since they express defective UGT family 1 isoenzymes and are also prone to acetaminophen toxicity. Acetaminophen kinetics were biphasic in all instances with distinct high and low affinity components. Km values for the high affinity activity in cat microsomes (0.31 +/- 0.1 mM; mean +/- SEM) were intermediate between those of dogs (0.11 +/- 0.02 mM) and humans (0.60 +/- 0.06 mM) and other species (0.22 to 6.7 mM; range). On the other hand, high affinity Vmax values were over 10-fold less in cat microsomes (0.025 +/- 0.006 nmol/min/mg) than in dogs (0.92 +/- 0.09 nmol/min/mg) and humans (0.27 +/- 0.09 nmol/min/mg); and over 5-fold less compared with microsomes from other species (range 0.13 to 7.63 nmol/min/mg). Gunn rat microsomes showed a similar 10-fold difference in high affinity Vmax values between the homozygous mutant (0.67 nmol/min/mg) and homozygous normal (6.75 nmol/min/mg) animals. These results demonstrate that, relative to a number of other species, cats have remarkably low hepatic levels of a high affinity acetaminophen-UGT. This difference is sufficient enough to explain poor glucuronidation of acetaminophen in vivo and susceptibility to acetaminophen intoxication.

Acetaminophen↗
PubMed1976

Computer simulation of leukemia therapy: combined pharmacokinetics, intracellular enzyme kinetics, and cell kinetics of the treatment of L1210 leukemia by cytosine arabinoside.

An integrated mathematic computer-based model of the pharmacokinetics, intracellular enzyme kinetics, and cell kinetics of the treatment of L1210 leukemia by cytosine arabinoside (ara-C) is described. The compartment model of Bischoff and Dedrick is extended to the intracellular level by inclusion of equations describing the phosphorylation, dephosphorylation, and deamination of ara-C with enzymatic feedback control. The activities of kinase, deaminase, and phosphatase are explicitly included in the models and are estimated from relevant data. Cell proliferation is described by a continuous-flow mathematic model in which cellular maturation and cell-to-cell variability in maturation rates are key variables. Cell proliferation is related to intracellular biochemistry through mathematic expressions which relate cell lethality and progression delay to the time course of intracellular ara-CTP. In vitro and in vivo experiments performed in a number of laboratories are compared by simulation. The most sensitive parameters in dose-response and cell-survival simulations are deoxycytidine kinase activity, ara-CTP half-life, renal clearance of ara-C, and cell-kinetic parameters for proliferation and cell killing. Progression delay is vital to the realistic simulation of divided-dose schedules. By comparative simulation we have identified areas of uncertainty which can be classified by a few additional measurements. The applications of simulations combining pharmacokinetic, biochemical, and cell-kinetic data in vitro and in vivo are discussed, exploring consistency among different measurements, and relating experimental protocols to clinical treatment.

Animals↗
PubMed1990

Enzyme kinetics and metabolic control. A method to test and quantify the effect of enzymic properties on metabolic variables.

It is usual to study the sensitivity of metabolic variables to small (infinitesimal) changes in the magnitudes of individual parameters such as an enzyme concentration. Here, the effect that a simultaneous change in all the enzyme concentrations by the same factor alpha (Co-ordinate-Control Operation, CCO) has on the variables of time-dependent metabolic systems is investigated. This factor alpha can have any arbitrary large value. First, we assume, for each enzyme measured in isolation, the validity of the steady-state approximation and the proportionality between reaction rate and enzyme concentration. Under these assumptions, any time-invariant variable may behave like a metabolite concentration, i.e. S alpha = Sr (S-type), or like a flux, i.e. J alpha = alpha Jr (J-type). The subscripts r and alpha correspond to the values of the variable before and after the CCO respectively. Similarly, time-dependent variables may behave according to S alpha (t/alpha) = Sr (t) (S-type) or to J alpha (t/alpha) = alpha J r (t) (J-type). A method is given to test these relationships in experimental systems, and to quantify deviations from the predicted behaviour. A positive test for deviations proves the violation of some of the assumptions made. However, the breakdown of the assumptions in an enzyme-catalysed reaction, studied in isolation, may or may not affect significantly the behaviour of the system when the component reaction is embedded in the metabolic network.

Analysis of Variance↗
PubMed1991

Dissociative inhibition of dimeric enzymes. Kinetic characterization of the inhibition of HIV-1 protease by its COOH-terminal tetrapeptide.

Human immunodeficiency virus 1 (HIV-1) protease is an aspartyl protease composed of two identical protomers linked by a four-stranded antiparallel beta-sheet consisting of the NH2- and COOH-terminal segments (Weber, I.T. (1990) J. Biol. Chem. 265, 10492-10496). Kinetic analysis of the HIV-1 protease-catalyzed hydrolysis of a fluorogenic substrate demonstrates that the enzyme is an obligatory dimer. At pH = 5.0, 0.1 M sodium acetate, 1 M NaCl, 1 mM EDTA buffer, 37 degrees C, the equilibrium dissociation constant, Kd = 3.6 +/- 1.9 nM. We found that the tetrapeptide Ac-Thr-Leu-Asn-Phe-COOH, corresponding to the COOH-terminal segment of the enzyme, is an excellent inhibitor of the enzyme. Kinetic analysis shows that the inhibitor binds to the inactive protomers and prevents their association into the active dimer (dissociative inhibition). The dissociative nature of this inhibition is consistent with the results obtained from sedimentation equilibrium experiments in which the apparent molecular weight of the enzyme was observed to be 20,800 +/- 1,500 and 12,100 +/- 300, in the absence and presence of the COOH-terminal tetrapeptide, respectively. The dissociation constant of the protomer-inhibitor complex is Ki = 45.1 +/- 1.8 microM. This is the first kinetic analysis and direct experimental demonstration of noncovalent dissociative inhibition.

Amino Acid Sequence↗
PubMed1989

Mechanism and binding specificity of beta-glucosidase-catalyzed hydrolysis of cellobiose analogues studied by competition enzyme kinetics monitored by 1H-NMR spectroscopy.

The application of high-resolution 1H-NMR spectroscopy to monitor substrate and product time dependencies in progress curve enzyme kinetics is described with beta-glucosidase-catalyzed hydrolyses of cellobiose analogues as examples. It is demonstrated that inhibition patterns, relative binding specificities and catalytic rates can be inferred from competition experiments with two or more substrates. It could be concluded from competition experiments that substrates which form less stable enzyme-substrate complexes than methyl beta-cellobioside are hydrolyzed faster than this reference substrate when they are the sole substrate, due to a lower activation energy in the catalytic step, but that they are hydrolyzed slower than the reference compound in direct competition, due to the formation of the less stable enzyme-substrate complex in the binding step.

Aspergillus niger↗
PubMed2000

Prediction of interindividual variation in drug plasma levels in vivo from individual enzyme kinetic data and physiologically based pharmacokinetic modeling.

A strategy is presented to predict interindividual variation in drug plasma levels in vivo by the use of physiologically based pharmacokinetic modeling and human in vitro metabolic parameters, obtained through the combined use of microsomes containing single cytochrome P450 enzymes and a human liver microsome bank. The strategy, applied to the pharmaceutical compound (N-[2-(7-methoxy-1-naphtyl)-ethyl]acetamide), consists of the following steps: (1) estimation of enzyme kinetic parameters K(m) and V(max) for the key cytochrome P450 enzymes using microsomes containing individual P450 enzymes; (2) scaling-up of the V(max) values for each individual cytochrome P450 involved using the ratio between marker substrate activities obtained from the same microsomes containing single P450 enzymes and a human liver microsome bank; (3) incorporation into a physiologically based pharmacokinetic model. For validation, predicted blood plasma levels and pharmacokinetic parameters were compared to those found in human volunteers: both the absolute plasma levels as well as the range in plasma levels were well predicted. Therefore, the presented strategy appears to be promising with respect to the integration of interindividual differences in metabolism and prediction of the possible impact on plasma and tissue concentrations of drugs in humans.

Acetamides↗
PubMedDate not supplied

Sex difference and possible relationship to microvascular complications of serum prorenin levels in type 2 diabetic patients, measured by a novel antibody-activating direct enzyme kinetic assay.

Prorenin, a precursor of renin, was measured in sera from normal subjects and type 2 diabetic patients, using a newly developed antibody-activating direct enzyme kinetic assay. Levels of prorenin were examined in relationship to diabetic microangiopathies. The levels of antibody-activating direct prorenin (AAD-PR) were approximately 1.5-fold higher than levels of prorenin measured by a conventional enzyme-activating method. AAD-PR levels were significantly higher in males than in females and in diabetic patients than in normal subjects. Moreover, AAD-PR levels were higher in diabetic patients with microalbuminuria and even higher in those with macroalbuminuria. In normoalbuminuric diabetic patients, AAD-PR levels were higher in those with retinopathy. Furthermore, a significant positive correlation was seen between the AAD-PR levels and HbA(lc) in normoalbuminuric diabetic subjects without retinopathy. Thus, the determination of circulating serum prorenin measured as AAD-PR is related to glycemia and in type 2 diabetic patients may be a risk marker of diabetic microangiopathy. More studies are necessary to determine whether AAD-PR may actually predict the development of microangiopathy.

Adult↗
PubMed1983

Estimates and implications of co-operativity for enzyme kinetics in the intact liver: method of flow invariants.

For substrates rapidly equilibrated between blood and liver cells, steady-state co-operative enzyme kinetics determines combinations of inlet and outlet substrate concentrations which do not change with the rate of blood flow recirculating through an isolated perfused liver. The mathematical forms of these combinations (here called flow invariants) are different for each value of the Hill co-operativity constant which can therefore be estimated, on a set of intact perfused preparations, from that flow invariant which is stochastically least dependent on experimental changes in the flow rate. This estimate, made in a narrow range of substrate concentrations, is illustrated using previously published data on the phosphorylation of galactose by galactokinase in rat liver. Changes (if any) of hepatocellular Hill constants in liver disease could be of clinical interest. A conspicuous difference between effects of negative and positive cooperativity in the intact perfused organ is found: for negative (but not for positive) co-operativity, complete extraction of the substrate in a single pass through a shunt-free liver is predicted from Hill's equation for a specified range of finite input concentrations and flow rates. Substrates with negative co-operativity in vivo would therefore facilitate the quantification of intrahepatic shunts.

Animals↗
PubMed1999

Enzyme kinetic characterization of the smooth muscle myosin phosphorylating system: activation by calcium and calmodulin and possible inhibitory mechanisms of antagonists.

A native-like smooth muscle filamentous myosin system was characterized from an enzyme kinetic point of view. The system contains endogenous myosin light chain kinase (MLCKase) and calmodulin (CM) (A. Sobieszek, J. Muscle Res. Cell Motil. 11 (1990) 114-124) and is, therefore, well suited for testing the action of CM-antagonists or other inhibitory compounds. However, this has not been done due to its complexity. The characterization of the system includes: (1) derivation of a relationship for rate of myosin phosphorylation in terms of total CM, free Ca2+ and total MLCKase concentrations, which includes only three binding constants; and (2) derivation of relationships between fractional inhibition rate (vi/vo) and total inhibitor concentration (It) which cover most of the inhibitory mechanisms applicable to the myosin system or to other CM-dependent enzymes. The three binding constants were subsequently evaluated from experimental data for filamentous myosin and for its isolated regulatory light chain (ReLC) using a non-linear regression software. They indicated differences in the interaction of myosin filament with the active CM-MLCKase complex in comparison to that of the isolated ReLC. The derived vi/vo versus It relationships, together with the software, make it possible to evaluate the inhibition constants and binding stoichiometries of CM-antagonists and other compounds inhibiting myosin phosphorylation. This approach was successfully applied to experimental data on inhibition of MLCKase by amiloride, cadmium, or CM-binding peptide (M-12) for simple mechanisms. For more complex mechanisms, inhibition by calmidozolium, trifluoperazine or melittin, the analysis showed that only calmidozolium acted specifically at the CM level in a multiple-site activator-depletion mechanism. Melittin and trifluoperazine inhibited the phosphorylation rate by a novel substrate-and-activator depletion mechanism, in which additional inhibition of the substrate resulted in the removal of the inhibition at lower range of the antagonists' concentration.

Animals↗
PubMed1979

The use of an oscillating-tube densitometer as a tool in enzyme kinetics. Determination of the influence of sodium ascorbate on invertase, dextransucrase and dextranase.

The use of a commercial oscillating-tube densitometer with an accuracy of 4 . 10(-7) g/cm3 for the determination of enzyme-kinetics constants is tested. This method is applied to the investigation of the influence of vitamin C (sodium ascorbate) on the glycolytic enzymes invertase, dextransucrase and dextranase. Invertase is inhibited uncompetitively, dextransucrase non-competitively. There is no significant effect of the vitamin on dextranase. The comparison of the mechanisms of the three enzymes suggests that only those reaction steps are inhibited by vitamin C in which fructose is released from the enzyme.

Ascorbic Acid↗