scholarly journals The probability that complex enzyme kinetic curves can be caused by activators or inhibitors

1981 ◽  
Vol 195 (3) ◽  
pp. 589-601 ◽  
Author(s):  
Francisco Solano-Muñoz ◽  
William G. Bardsley ◽  
Keith J. Indge
Keyword(s):  
Rate Equation ◽  
Kinetic Scheme ◽  
Single Step ◽  
Enzyme Kinetic ◽  
Curve Shape ◽  
Implicit Representation ◽  
Complex Curves ◽  
Allosteric Effectors ◽  
S Curve ◽  
Computer Studies

Numerous chemical compounds are known that alter the rate of conversion of substrates into products in enzyme-catalysed reactions by interacting with the enzyme rather than substrates. Where this takes place in such a way that the effect is reversible on removing the compound, say by dialysis, and where the compound is unchanged chemically by the enzyme system, we refer to such a compound as a modifier. So protons, inorganic salts, activators, inhibitors or even specific allosteric effectors would all be modifiers, and any chemically reasonable kinetic scheme that is proposed to account for such effects is referred to as modifier mechanism. Three versions of a modifier mechanism of enzyme action are studied. The implicit representation is 2:2 in [S] (with α0=0) and 2:2 in [M] (with α0≠0), and this is a short-hand scheme for the minimum chemical formulation, the explicit one, involving discrete ES and EP species, which is 2:2 in [S] (with α0=0) and 3:3 in [M] (with α0≠0). If m extra steps are allowed between interconversion of ES and EP species, the degree of the rate equation remains 2:2 in [S] (with α0=0), but increases to degree (m+3):(m+3) in modifier (with α0≠0). It is proved that this increase in degree is genuine and that highly complex v([M]) (i.e. v-versus-[M]) curves can occur. Computation of the probabilities of the five possible double-reciprocal plots in 1/v versus 1/[S] show that all of these formulations of the modifier mechanism give similar probabilities, and these are characteristic for the mechanism and quite distinct from the intrinsic curve-shape probabilities. It is also established that the probabilities of alternative complex v([M]) plots are similar for the various formulations, and again the probabilities of the allowed complex curves for the mechanism are quite distinct from the instrinsic probabilities of the ten possible v([M]) curves for a 2:2 function (with α0≠0). The computer studies reported lead to several conclusions about the probability of modifiers leading to inhibition or activation or causing changes in v([S]) curve shapes, and suggest that differentiation between model mechanisms may be facilitated by knowledge of the intrinsic curve-shape probabilities for the appropriate degree rational function and the characteristic way that this is altered by specific mechanisms. It is shown that, although in some instances new curve-shape complexities are possible when schemes are considered that allow for interconversion of ES and EP species, these are highly improbable and, for theoretical purposes, schemes formulated with node compression provide good approximations to the more complicated explicit schemes. By node compression we refer to the procedure whereby enzyme kinetic schemes are simplified by replacing sequences of steps such as ES⇌X1⇌X2⇌...⇌EP... by a single step... ES/EP... that does not formally recognize the existence of the intermediate species. We show that the modifier mechanism studied is one where this process alters the form of the rate equation.

scholarly journals The quantitative analysis of ligand binding and initial-rate data for allosteric and other complex enzyme mechanisms

1976 ◽  
Vol 153 (1) ◽  
pp. 101-117 ◽  
Author(s):  
W G Bardsley
Keyword(s):  
Kinetic Data ◽  
Quantitative Data ◽  
Initial Rate ◽  
Graphical Methods ◽  
Enzyme Kinetic ◽  
Rate Data ◽  
Curve Shape ◽  
High Substrate Concentration ◽  
Complex Curve ◽  
Complex Curves

1. The eight methods for plotting enzyme kinetic data are classified and analysed, and it is shown how, in each case, it is only possible to obtain quantitative data on the coefficients of the lowest- and highest-degree terms in the rate equation. 2. The combinations of coefficients that are accessible experimentally from limiting slopes and intercepts at both low and high substrate concentration are stated for all the graphical methods and the precise effects of these on curve shape in different spaces is discussed. 3. Ambiguities arising in the analysis of complex curves and certain special features are also investigated. 4. Four special ordering functions are defined and investigated and it is shown how knowledge of these allows a complete description of all possible complex curve shapes.

PLATELET AGGREGATION DOES NOT CONFORM TO SIMPLE PARTICLE COLLISION THEORY

1987 ◽  
Author(s):  
Moideen P Jamaluddin
Keyword(s):  
Platelet Aggregation ◽  
Rate Equation ◽  
Rate Constants ◽  
Kinetic Scheme ◽  
Order Type ◽  
Second Order ◽  
Particle Collision ◽  
Collision Theory ◽  
Rate Law ◽  
First Order

Platelet aggregation kinetics, according to the particle collision theory, generally assumed to apply, ought to conform to a second order type of rate law. But published data on the time-course of ADP-induced single platelet recruitment into aggregates were found not to do so and to lead to abnormal second order rate constants much larger than even their theoretical upper bounds. The data were, instead, found to fit a first order type of rate law rather well with rate constants in the range of 0.04 - 0.27 s-1. These results were confirmed in our laboratory employing gelfiltered calf platelets. Thus a mechanism much more complex than hithertofore recognized, is operative. The following kinetic scheme was formulated on the basis of information gleaned from the literature.where P is the nonaggregable, discoid platelet, A the agonist, P* an aggregable platelet form with membranous protrusions, and P** another aggregable platelet form with pseudopods. Taking into account the relative magnitudes of the k*s and assuming aggregation to be driven by hydrophobic interaction between complementary surfaces of P* and P** species, a rate equation was derived for aggregation. The kinetic scheme and the rate equation could account for the apparent first order rate law and other empirical observations in the literature.

scholarly journals Deviations from Michaelis-Menten kinetics. The possibility of complicated curves for simple kinetic schemes and the computer fitting of experimental data for acetylcholinesterase, acid phosphatase, adenosine deaminase, arylsulphatase, benzylamine oxidase, chymotrypsin, fumarase, galactose dehydrogenase, β-galactosidase, lactate dehydrogenase, peroxidase and xanthine oxidase

1980 ◽  
Vol 187 (3) ◽  
pp. 739-765 ◽  
Author(s):  
W G Bardsley ◽  
P Leff ◽  
J Kavanagh ◽  
R D Waight
Keyword(s):  
Experimental Data ◽  
Rate Equation ◽  
Substrate Inhibition ◽  
Rate Equations ◽  
Double Reciprocal Plot ◽  
Binding Model ◽  
Steady State Rate ◽  
Benzylamine Oxidase ◽  
S Curve ◽  
Galactose Dehydrogenase

The possible graph shapes for one-site/two-state and substrate-modifier models are discussed. The two-state model is a version of the Monod-Wyman-Changeux model and gives a rate equation with 240 denominator terms. Discussion in terms of K and V effects is not possible. A simplified version of the mechanism can be shown to give v-versus-[S] curves that are either sigmoid or non-sigmoid. They may show substrate inhibition or no final maximum, and the double-reciprocal plots can be concave up or down. The corresponding binding model is determined by only two constants and gives a linear double-reciprocal plot. The substrate-modifier mechanism is a simple example of a mechanism where inclusion of catalytic steps leads to a genuine increase in degree of the rate equation. The v-versus-[S] curve can show such complexities as two maxima and a minimum, and the double-reciprocal plot can cross its asymptote twice, proving the rate equation to be 4:4. A simplified version is 3:3, and analysis shows that at least 18 of the 27 double-reciprocal plots that can arise with 3:3 functions are possible with this particular mechanism. Representative double-reciprocal and Scatchard plots are presented for several sets of rate-constant values. It is concluded that relatively simple mechanisms give pseudo-steady-state rate equations of high degree and considerable complexity. With extended ranges of substrate concentrations there is every reason to believe that experimental data would show the sort of deviations from Michaelis-Menten kinetics seen with calculated curves for such simple mechanisms. Narrow ranges of substrate concentration, on the other hand, would lead to inflexions and curvature being overlooked. It is not possible to discuss such deviations from Michaelis-Menten kinetics in terms of kinetic constants such as Km and V, and, in general, it is also difficult to see any simple way to explain intuitively such features as sigmoidicity, substrate inhibition, double-reciprocal convexity and decrease in degree by cancellation of common factors between numerator and denominator of rate equations. These conclusions apply with even more force when catalytic steps are included, for then the rate equations, are for multi-site mechanisms, of higher degree, allowing increasingly complex curve shapes. A number of enzymes were studied and initial-rate data were fitted by computer.(ABSTRACT TRUNCATED AT 400 WORDS)

scholarly journals Luminometric single step urea assay using ATP-hydrolyzing urease

1998 ◽  
Vol 44 (9) ◽  
pp. 1964-1973 ◽  
Author(s):  
Birgitta Näslund ◽  
Lars Ståhle ◽  
Arne Lundin ◽  
Björn Anderstam ◽  
Peter Arner ◽  
...  
Keyword(s):  
Multivariate Analysis ◽  
Biological Fluids ◽  
Single Step ◽  
Enzyme Kinetic ◽  
Spectrophotometric Methods ◽  
Analytical Interference ◽  
Dependent Activity ◽  
Highly Correlated ◽  
Assay Conditions

Abstract An automatic enzyme kinetic luminometric method for determination of small quantities of urea in biological fluids and in microdialysates is presented. The method is based on the ATP-hydrolyzing urease reaction [urea amidohydrolase (ATP-hydrolyzing); EC 3.5.1.45], monitored by a luciferin-luciferase ATP reaction. The assay range is 100 pmol to 50 nmol with a detection limit of 5 μmol/L in the sample, compared with detection limits of 0.1 mmol/L in earlier spectrophotometric methods. To reduce the non-urea-dependent ATPase activity (vblank) and to increase the urea-dependent activity, 1,2-propanediol was included. Assay conditions were optimized by multivariate analysis. Recoveries of urea added to blood dialysate and plasma were 96–103%. No analytical interference of common metabolites, drugs, or other additives was observed. The total CVs (6 days and six concentrations, 1.2–21.8 mmol/L) were 3.6–8.5%. The results obtained with the present assay were highly correlated for dialysate (r = 0.979) and for plasma (r = 0.978) with those obtained by a spectrophotometric kit method with slopes of 1.02–1.03 and intercepts of 0.08–0.23 mmol/L.

scholarly journals Deviations from Michaelis–Menten kinetics. Computation of the probabilities of obtaining complex curves from simple kinetic schemes

1981 ◽  
Vol 193 (1) ◽  
pp. 339-352 ◽  
Author(s):  
F Solano-Muñoz ◽  
P B McGinlay ◽  
R Woolfson ◽  
W G Bardsley
Keyword(s):  
Probability Distributions ◽  
Rational Functions ◽  
Distribution Functions ◽  
Rate Equations ◽  
British Library ◽  
Curve Shape ◽  
Product Release ◽  
Complex Curves ◽  
Probability Distribution Functions ◽  
Approximate Magnitude

1. It is possible to calculate the intrinsic probability associated with any curve shape that is allowed for rational functions of given degree when the coefficients are independent or dependent random variables with known probability distributions. 2. Computations of such probabilities are described when the coefficients of the rational function are generated according to several probability distribution functions and in particular when rate constants are varied randomly for several simple model mechanisms. 3. It is concluded that each molecular mechanism is associated with a specific set of curve-shape probabilities, and this could be of value in discriminating between model mechanisms. 4. It is shown how a computer program can be used to estimate the probability of new complexities such as extra inflexions and turning points as the degree of rate equations increases. 5. The probability of 3 : 3 rate equations giving 2 : 2 curve shapes is discussed for unrestricted coefficients and also for the substrate-modifier mechanisms. 6. The probability associated with the numerical values of coefficients in rate equations is also calculated for this mechanism, and a possible method for determining the approximate magnitude of product-release steps is given. 7. The computer programs used in the computations have been deposited as Supplement SUP 50113 (21 pages) with 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. (1978) 169, 5.

V884: Demonstration of a Novel Single-Step Percutaneous Access Sheath for Nephrostolithotomy: A Video Presentation

2005 ◽  
Vol 173 (4S) ◽  
pp. 240-240
Author(s):  
Premal J. Desai ◽  
David A. Hadley ◽  
Lincoln J. Maynes ◽  
D. Duane Baldwin
Keyword(s):  
Single Step ◽  
Video Presentation ◽  
Percutaneous Access ◽  
Access Sheath

Effect of Lipoprotein(a) and LDL on Plasminogen Binding to Extracellular Matrix and on Matrix-dependent Plasminogen Activation by Tissue Plasminogen Activator

1996 ◽  
Vol 75 (03) ◽  
pp. 497-502 ◽  
Author(s):  
Hadewijch L M Pekelharing ◽  
Henne A Kleinveld ◽  
Pieter F C.C.M Duif ◽  
Bonno N Bouma ◽  
Herman J M van Rijn
Keyword(s):  
Extracellular Matrix ◽  
Endothelial Cells ◽  
Plasminogen Activator ◽  
Binding Sites ◽  
Umbilical Vein ◽  
Single Step ◽  
Plasminogen Activation ◽  
Structural Homology ◽  
Tissue Plasminogen ◽  
Umbilical Vein Endothelial Cells

SummaryLp(a) is an LDL-like lipoprotein plus an additional apolipoprotein apo(a). Based on the structural homology of apo(a) with plasminogen, it is hypothesized that Lp(a) interferes with fibrinolysis. Extracellular matrix (ECM) produced by human umbilical vein endothelial cells was used to study the effect of Lp(a) and LDL on plasminogen binding and activation. Both lipoproteins were isolated from the same plasma in a single step. Plasminogen bound to ECM via its lysine binding sites. Lp(a) as well as LDL were capable of competing with plasminogen binding. The degree of inhibition was dependent on the lipoprotein donor as well as the ECM donor. When Lp(a) and LDL obtained from one donor were compared, Lp(a) was always a much more potent competitor. The effect of both lipoproteins on plasminogen binding was reflected in their effect on plasminogen activation. It is speculated that Lp(a) interacts with ECM via its LDL-like lipoprotein moiety as well as via its apo(a) moiety.

Purification of Urokinase from Complex Mixtures Using Immobilized Monoclonal Antibody Against Urokinase Light Chain

1983 ◽  
Vol 49 (01) ◽  
pp. 024-027 ◽  
Author(s):  
David Vetterlein ◽  
Gary J Calton
Keyword(s):  
Monoclonal Antibody ◽  
Light Chain ◽  
Culture Media ◽  
Biological Fluids ◽  
Single Step ◽  
High Yield ◽  
Step Method ◽  
Commercial Preparation ◽  
E Coli ◽  
Tissue Culture Media

SummaryThe preparation of a monoclonal antibody (MAB) against high molecular weight (HMW) urokinase light chain (20,000 Mr) is described. This MAB was immobilized and the resulting immunosorbent was used to isolate urokinase starting with an impure commercial preparation, fresh urine, spent tissue culture media, or E. coli broth without preliminary dialysis or concentration steps. Monospecific antibodies appear to provide a rapid single step method of purifying urokinase, in high yield, from a variety of biological fluids.

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