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D J Winzor

Publications and source records attributed to D J Winzor.

At least 91 records · Page 5Linked to original sources
PubMed1989

Effect of calcium ion on the interaction of aldolase with rabbit muscle myofibrils.

A partition equilibrium study has shown calcium ion to be a noncompetitive inhibitor of aldolase adsorption by rabbit muscle myofibrils. This inhibition is interpreted quantitatively in terms of a 10-fold decrease in the intrinsic association constant for the aldolase-myofibril interaction upon Ca2+ binding to either or both of the low-affinity troponin sites associated with regulation of muscle contraction.

Animals↗
PubMed1989

Thermodynamic nonideality in enzyme catalysis. Effect of albumin on the reduction of pyruvate by lactate dehydrogenase.

The enhanced catalytic reduction of pyruvate by rabbit muscle lactate dehydrogenase that results from the addition of serum albumin [Nichol, L. W., Sculley, M. J., Ward, L. D. & Winzor, D. J. (1983) Arch. Biochem. Biophys. 222, 574-581] is shown to emanate solely from an increase in maximal velocity, there being no discernible effect of this inert space-filling macromolecular solute on the Michaelis constant for either pyruvate or NADH. As part of the search for a mechanistic explanation of this kinetic phenomenon, the space-filling effects of albumin have been used to eliminate the possibility that the increase in sedimentation coefficient of lactate dehydrogenase effected by inclusion of oxamate with enzyme-NADH complex reflects preferential binding of this pyruvate analog to a more compact isomeric state of the binary complex. The enzyme kinetic results are therefore considered in terms of a reaction scheme entailing gross conformational changes during the formation of ternary enzyme-NADH-pyruvate complex and its isomerization to an activated transition state. The experimentally observed insensitivity of the Michaelis constant for pyruvate to albumin concentration is in keeping with theoretical prediction, but incorporation of the measured extent of maximal velocity enhancement into the kinetic model leads to a predicted volume for the fully saturated transition-state complex that is far too small to be experimentally feasible. A more complex mechanistic model involving additional isomerizations of enzyme-substrate species is thus required to achieve quantitative description of the albumin effect solely in terms of thermodynamic nonideality.

Kinetics↗
PubMed1989

Biospecific interactions: their quantitative characterization and use for solute purification.

Biospecificity is due largely to the formation and dissociation of non-covalent complexes between small molecules and macromolecules, or between two macromolecules. The first part of this review is concerned with the use of such biospecificity in the fractionation and identification of solutes. Major emphasis is given to affinity chromatography, especially in regard to the practical considerations inherent in an experimental situation and to the wide range of specific interactions that can be utilized. The second part of the review considers the quantitative characterization of biospecific complex formation. The merits of frontal gel chromatography, electrophoretic methods and affinity chromatography are discussed, and special consideration is given to the effects of ligand and/or acceptor multivalency because of its relevance to many biospecific interactions. Finally attention is drawn to the feasibility of employing quantitative affinity chromatographic theory for the determination of association constants for antigen-antibody systems by radioimmunoassay and enzyme-linked immunosorbent assay techniques.

Biochemistry↗
PubMed1989

Space-filling effects of inert solutes as probes for the detection and study of substrate-mediated conformational changes by enzyme kinetics: theoretical considerations.

From expressions derived for the space-filling effects of small inert solutes on kinetic parameters for univalent enzymes undergoing isomerizations that are substrate-induced and pre-existing, it is concluded that experimental observation of an enhanced maximal velocity in the presence of inert solute can only reflect the existence of the former type of conformational change; and that the isomerization must be governed by a relatively small equilibrium constant. Similar conclusions apply to multivalent enzymes exhibiting Michaelis-Menten kinetics. Extension of the theory to provide quantitative expressions for multivalent enzymes has made possible the numerical simulation of thermodynamic non-ideality effects on systems conforming with the Monod and Koshland models of allostery. In that regard the simulated Scatchard plots for the two models differ sufficiently in form to suggest that detailed examination of the space-filling effects of small solutes on the kinetics of an allosteric enzyme may, under favourable circumstances, allow identification of the appropriate allosteric mechanism. Finally, these considerations of thermodynamic non-ideality in relation to the kinetics of allosteric enzymes have revealed formal similarities between the consequences of space-filling by inert solutes and the specific effects of allosteric activators or inhibitors. Attention is drawn to the possible implications of this observation in relation to the functioning of allosteric enzymes in vivo, where catalytic performance may be modified by factors no more specific than the ability of unrelated solutes to occupy space in the highly concentrated cellular environment.

Chemical Phenomena↗
PubMed1989

Numerical and experimental demonstrations of the need for caution in the use of zonal gel chromatography for characterizing ligand interactions with small acceptors.

Quantitative expressions are presented for the evaluation of equilibrium constants for interactions of the type A + B in equilibrium C from experiments entailing the application of a small zone of acceptor-ligand mixture to a column of gel preequilibrated with ligand solution [J.P. Hummel and W.J. Dreyer (1962) Biochim. Biophys. Acta 63, 530-532]. Only in the event that identical elution volumes pertain to acceptor and complex does the steady-state binding constant (Kss) obtained by that method equal the thermodynamic equilibrium constant (K). Simulated elution profiles are then generated with parameters relevant to gel chromatography of the ATP-Mg2+ system on Sephadex G-10 in order to demonstrate the practical importance of the need for distinction between Kss and K in situations where acceptor and complex do not comigrate. A study of the interaction between soybean trypsin inhibitor and cytochrome c by gel chromatography on Sephadex G-75 is then used to illustrate the feasibility of combining information from Hummel-Dreyer experiments with the theoretical expressions to characterize systems under the more general conditions that the elution volumes of A and C differ. A finding of considerable theoretical interest in relation to the simulation of mass migration behavior is the demonstration that a truncation error is the source of zonal spreading in the theoretical-plate model of chromatography. This truncation error is shown to be the source of spreading generated whenever solution of an abbreviated (diffusion-free) continuity equation involves substituting first differences for first derivatives in the differential equation describing mass transport.

Adenosine Triphosphate↗
PubMed1989

Effects of thermodynamic nonideality on the kinetics of ester hydrolysis by alpha-chymotrypsin: a model system with preexistence of the isomerization equilibrium.

The effects of a small inert solute, sucrose, on the kinetics of hydrolysis of N-acetyl-tryptophan ethyl ester by bovine alpha-chymotrypsin have been investigated. In studies at pH 7 and 20 degrees C the presence of 0.5 M sucrose in assay mixtures caused no discernible change in kinetic parameters, a result consistent with existence of the enzyme in a single conformational state under those conditions. However, at pH 3.5 and 50 degrees C, conditions under which the enzyme comprises an equilibrium mixture of compact and expanded isomeric states, inclusion of the inert solute led to a considerable decrease in Michaelis constant (0.84 to 0.61 mM) but no significant change in maximal velocity. These results were shown to be amenable to quantitative interpretation in terms of thermodynamic nonideality effects on catalysis by an enzyme undergoing reversible isomerization in the absence of substrate. For that analysis, which required experimental estimates of the equilibrium constant for preexisting isomerization of enzyme and the activity coefficient of substrate, the magnitude of the former (0.3) was obtained by difference spectroscopy: liquid-liquid partition studies with bromobenzene as organic phase were used to determine the effect of sucrose on the activity coefficient of N-acetyltryptophan ethyl ester. Such agreement between experimental kinetic findings and theoretical predictions based on considerations of excluded volume points to the possible use of the space-filling effects of small solutes for delineating the gross extent of conformational changes associated with reversible isomerization of proteins, and hence to the potential of thermodynamic nonideality as a probe for studying protein denaturation mechanisms as well as substrate-mediated changes associated with enzyme reaction mechanisms.

Chymotrypsin↗
PubMed1989

Evaluation of equilibrium constants from precipitin curves: interaction of alpha-crystallin with an elicited monoclonal antibody.

A simple procedure, based on the precipitin curve and the antibody:antigen ratios of the precipitates, is described for evaluation of the intrinsic association constant (k) governing the interaction between a multivalent antigen and a bivalent antibody. Its application is illustrated with a study of the interaction between alpha-crystallin and an elicited monoclonal antibody, which is shown to exhibit essentially identical affinities (k = 9 X 10(4) M-1) for the alpha m and alpha c forms of the antigen.

Animals↗
PubMed1989

Evaluation of nonideality from gel chromatographic partition coefficients. A technique with greater versatility than equilibrium dialysis.

Frontal gel chromatography has been used to measure partition coefficients which enable a quantitative evaluation of the thermodynamic nonideality of small solutes generated by the presence of high concentrations of macromolecular solutes. Equivalence of results obtained by the present method and by equilibrium dialysis is demonstrated in a comparison of results for dextran sulfate-NaCl and dextran-sorbitol systems. Interaction coefficients obtained for dextran-sorbitol and protein-polyethylene glycol 4000 systems yields results which are in reasonable agreement with those predicted on the statistical-mechanical basis of excluded volume. Because of its greater versatility in regard to the range of systems that may be studied, the frontal gel chromatographic procedure is likely to be of particular value for the quantitative characterization of thermodynamic nonideality arising from excluded volume effects in concentrated mixtures of macromolecular solutes.

Biopolymers↗
PubMed1988

Allowance for effects of electrostatic repulsion on protein dimerization.

A simple procedure for assessing the extent of electrostatic effects on protein dimerization is described and illustrated by application to published results on the ionic strength dependence of the dimerization constant for alpha-chymotrypsin at pH 4 (Aune, K.C., Goldsmith, L.C. and Timasheff, S.N. (1971) Biochemistry 10, 1617-1622). From the analysis it is concluded that the inverse dependence of alpha-chymotrypsin dimerization upon ionic strength is predominantly a general electrostatic effect, rather than a consequence of repulsion between two specific charged residues on the adjacent monomers comprising dimer.

Chemical Phenomena↗
PubMed1988

Substrate as a source of thermodynamic nonideality in enzyme kinetic studies: invertase-catalyzed hydrolysis of sucrose.

Expressions for the effects of thermodynamic nonideality arising from the use of high concentrations of small substrate in enzyme kinetic studies are derived. Their application to experimental results for the hydrolysis of sucrose by yeast invertase (pH 4.9, 37 degrees C) signifies that the progressive decrease in initial velocity at high sucrose concentration is consistent with the occurrence of isomeric expansion during the transition of an enzyme-substrate complex to its activated state. Ultracentrifuge studies on the yeast enzyme preparation are then used to establish the physical acceptability of the volume change required to account for the kinetic effects in these terms: the postulated expansion of 1.3 liter/mol would represent a mere 0.16% increase in hydrated volume (or a corresponding increase in extent of asymmetry). Finally, although originally interpreted to signify an effect of sucrose on water concentration, published results for the invertase-sucrose system [J. M. Nelson and M. P. Schubert (1928) J. Amer. Chem. Soc. 50, 2188-2193] also find a rational explanation in terms of the present analysis based on effects of thermodynamic nonideality in enzyme kinetic studies.

Catalysis↗
PubMed1988

Direct experimental evidence for competitive inhibition of dihydrofolate reductase by methotrexate.

Steady-state enzyme kinetic techniques at very low enzyme concentration (0.4 nM) were used successfully to measure the inhibition constant (53 pM) for the dissociation of methotrexate from the ternary complex of methotrexate, NADPH and dihydrofolate reductase from Lactobacillus casei; and to demonstrate unequivocally that the inhibition was, indeed, competitive with respect to dihydrofolate.

Binding, Competitive↗
PubMed1988

Thermodynamic characterization of the interactions of methotrexate with dihydrofolate reductase by quantitative affinity chromatography.

Affinity chromatography on methotrexate-Sepharose has been used to evaluate dissociation constants for interactions of methotrexate with dihydrofolate reductase from Lactobacillus casei. Equilibrium constants of 0.25 microM and 0.6 nM were obtained for dissociation of the inhibitor from the enzyme-methotrexate and enzyme-NADPH-methotrexate complexes, respectively, these estimates being in good agreement with the corresponding published values for dihydrofolate reductase from Streptococcus faecium. By employing a different method for evaluating the thermodynamic dissociation constant for the enzyme-NADPH-methotrexate interaction, this investigation provides independent support for the inference drawn from published fluorescence quenching studies that the interaction of methotrexate with dihydrofolate reductase-NADPH complex is governed by a dissociation constant in the vicinity of 600 pM, a value slightly higher than, but approaching, the inhibition constant of 50-60 pM obtained by enzyme kinetic techniques.

Chromatography, Affinity↗
PubMed1988

Graphical analysis of binding data reflecting competition between two ligands for the same acceptor sites.

A theoretical expression is derived for the analysis of results from competitive binding studies in which two multivalent ligands compete for acceptor sites, and a linear transform is suggested for simple graphical representation and assessment of experimental results. The protocol is illustrated by application to competitive binding data, obtained by ultrafiltration, on the interactions of bovine serum albumin with two structurally similar organic anions, methyl orange and methyl red. In a second experimental study the present approach is then used to establish that lactate dehydrogenase and aldolase compete for the same myofibrillar sites of bovine cardiac muscle. Finally, numerically simulated behavior of systems with additional binding sites for either ligand is used to emphasize that the criterion for classical (complete) competition is agreement between an experimentally determined equilibrium constant for ligand binding and the apparent value deduced from competitive binding studies. Nevertheless, the present analysis of competitive binding data should still offer considerable scope for screening quantitatively the cross-reactivities of drug and antigen analogs for their respective specific protein-acceptor sites.

Animals↗
PubMed1988

Thermodynamic nonideality as a probe of allosteric mechanisms: preexistence of the isomerization equilibrium for rabbit muscle pyruvate kinase.

Sedimentation velocity studies in the presence and absence of an inert space-filling solute, sucrose, have been used to establish preexistence of the isomerization equilibrium responsible for the allosteric behavior of rabbit muscle pyruvate kinase. Whereas the inclusion of phenylalanine (5 mM) with enzyme gives rise to a decrease of 0.3 S in the sedimentation coefficient of pyruvate kinase, the corresponding effect of phosphoenolpyruvate is to increase the sedimentation coefficient by 0.03 S. Consideration of these findings to signify the existence of an isomeric equilibrium between compact and expanded forms of the enzyme is substantiated by the finding that inclusion of sucrose (0.1 M) also brings about the change in sedimentation coefficient effected by phosphoenolpyruvate. By demonstrating that rabbit muscle pyruvate kinase undergoes isomerization in the absence of substrate, this study removes any necessity to consider the existence of an isomerization equilibrium that is substrate-induced; and thereby provides experimental support for adoption of the Monod model of allostery to interpret enzyme kinetic data for pyruvate kinase [R. W. Oberfelder, B. G. Barisas, and J. C. Lee (1984) Biochemistry 23, 3822-3826].

Animals↗
PubMed1988

Comparison of paraquat-specific murine monoclonal antibodies produced by in vitro and in vivo immunization.

The techniques of in vitro and in vivo immunization have been used to produce murine hybridomas secreting anti-paraquat IgM and IgG antibodies, respectively. The monoclonal antibodies from these IgM-secreting and IgG-secreting cell lines have been purified by HPLC and characterized by ELISA for their affinities to eliciting antigen and cross-reactivity. Both antibody preparations exhibited high selectivity and similar affinities for paraquat. There was only a minor degree of interaction with a range of paraquat analogs that differed in the nature and position of group substituents.

Animals↗
PubMed1988

Effect of sucrose on the dimerization of alpha-chymotrypsin. Allowance for thermodynamic nonideality arising from the presence of a small inert solute.

The space-filling effects of sucrose on the dimerization of alpha-chymotrypsin have been investigated by sedimentation equilibrium studies on the enzyme in acetate-chloride buffer, pH 3.9, I 0.2. From the extent of enhancement of the apparent dimerization constant in the presence of 0.05-0.16 M sucrose, it is concluded that this effect of thermodynamic nonideality finds quantitative explanation in terms of excluded volume. However, the suggested approximation that the radius of an inert small solute would be sufficiently small to be neglected in the calculation of covolumes (D.J. Winzor and P.R. Wills, Biophys. Chem. 25 (1986) 243) has not withstood the more stringent test afforded by the present study of alpha-chymotrypsin dimerization. A value of 0.34 nm for the effective thermodynamic radius of sucrose was inferred from the covolume for self-interaction obtained by frontal gel chromatography on Sephadex G-10 under the conditions of the ultracentrifugal studies. Finally, results of sedimentation equilibrium experiments on alpha-chymotrypsin in the presence of 0.1 M glycerol were also shown to be consistent with interpretation in terms of the model of space-filling effects entailing complete exclusion of small solute from the hydrated protein domain.

Chymotrypsin↗
PubMed1988

Accumulation of ascorbate in rat cerebellum.

Ultrafiltration and gel chromatography on Sephadex G-75 have revealed no evidence of ascorbate-binding to protein components of the soluble fraction derived from a tissue homogenate of rat cerebellum. Furthermore, partition studies have failed to detect any binding of ascorbic acid to structural components of rat cerebellum, as the ascorbate content of cerebellar tissue exhibited linear dependence upon the concentration of free vitamin in the surrounding medium (0.1 M phosphate, pH 6.5). The accumulation of ascorbic acid in rat cerebellum is discussed, therefore, in terms of an elevated intracellular concentration maintained by active transport of the vitamin across cerebellar tissue membranes.

Animals↗