Biomedical subjects

D J Winzor

Publications and source records attributed to D J Winzor.

At least 73 records · Page 4Linked to original sources

PubMed1991

Preferential ligand binding to multi-state acceptor systems: comparisons of the calcium-binding and dimerization characteristics of prothrombin and fragment 1.

Consideration is given to the interactions of a ligand with self-associating acceptor systems for which preferential binding is an ambiguous term in that ligand-mediated self-association does not necessarily imply a greater binding constant for polymeric acceptor--even in instances where binding sites are preserved in the self-association process. This dilemma is shown to arise in situations involving the binding of ligand to monomeric and polymeric forms of an acceptor that also coexist in equilibrium with inactive isomeric states. For example, the ten-fold increase in the measured dimerization constant for prothrombin Fragment 1 in the presence of a saturating concentration of Ca2+ ion may well reflect the existence of a 12% greater binding constant for the interaction of metal ion with dimeric acceptor. However, that result, as well as the detailed form of the sigmoidal binding curve, are also reasonably described by another extreme model in which the monomeric and dimeric forms of the acceptor possess equal affinities for Ca2+ ion. Likewise, the fact that the same experimental dimerization constant applies to prothrombin and its Ca(2+)-saturated complex does not preclude the possibility that the active form of dimeric zymogen exhibits a 12% greater affinity for metal ion. Numerical simulations have established that characterization of the dimerization behaviour as a function of free ligand concentration should allow greater discrimination between such models of the interplay between calcium binding and self-association of prothrombin and Fragment 1. Finally, by illustrating the likelihood that the disparity in self-association behaviour of prothrombin and Fragment 1 merely reflects minor differences in the relative magnitudes of isomerization constants and/or binding constants for monomeric and dimeric states of the two acceptors, the present investigation serves to allay concern about the validity of employing the proteolytic fragment as a model of the intact zymogen.

Calcium↗

PubMed1991

Evaluation of antigen-antibody affinity constants by partition equilibrium studies with a two-phase aqueous polymer system: a more rigorous analysis.

A quantitative procedure is devised for the determination of affinity constants from the composition-dependence of the antigen partition coefficient in liquid-liquid partition studies of antigen-antibody mixtures. Its use is illustrated by application to published results (Elling, L.R., Kula, M.-R., Hadas, E. and Katchalski-Katzir, E. (1991) Anal. Biochem. 192, 74-77) obtained for the interaction of horseradish peroxidase with an elicited monoclonal antibody by antigen distribution studies with a two-phase aqueous polymer system (poly(ethylene glycol)/dextran) for the partition analysis. Shortcomings in the original interpretation of these results are eliminated.

Antibodies, Monoclonal↗

PubMed1991

Effects of thermodynamic nonideality in kinetic studies: evidence of an expanded intermediate complex in the inactivation of thrombin by antithrombin III.

The technique of competitive chromogenic substrate hydrolysis is used to examine the inhibitory effects of sucrose and glycerol on the inactivation of thrombin by antithrombin III. This inhibition is attributed to the existence of a slight increase in volume/asymmetry associated with formation of the thrombin-antithrombin complex that subsequently undergoes covalent modification in an irreversible inactivation step. Partial reversal of the equilibrium step is thus considered to result from the effects of molecular crowding in the highly concentrated environment that is generated by the inclusion of these small insert solutes.

Antithrombin III↗

PubMed1991

Use of quantitative affinity chromatography for characterizing high-affinity interactions: binding of heparin to antithrombin III.

The versatility of quantitative affinity chromatography (QAC) for evaluating the binding of macromolecular ligands to macromolecular acceptors has been increased substantially as a result of the derivation of the equations which describe the partitioning of acceptor between matrix-bound and soluble forms in terms of total, rather than free, ligand concentrations. In addition to simplifying the performance of the binding experiments, this development makes possible the application of the technique to systems characterized by affinities higher than those previously amenable to investigation by QAC. Addition of an on-line data acquisition system to monitor the concentration of partitioning solute in the liquid phase as a function of time has permitted the adoption of an empirical approach for determining the liquid-phase concentration of acceptor in the system at partition equilibrium, a development which decreases significantly the time required to obtain a complete binding curve by QAC. The application of these new QAC developments is illustrated by the determination of binding constants for the interactions of high-affinity heparin (Mr 20,300) with antithrombin III at three temperatures. Association constants of 8.0 +/- 2.2 x 10(7), 3.4 +/- 0.3 x 10(7), and 1.0 +/- 0.2 x 10(7) M-1 were observed at 15, 25, and 35 degrees C, respectively. The standard enthalpy change of -4.2 +/- 0.6 kcal/mol that is calculated from these data is in good agreement with a reported value obtained from fluorescence quenching measurements.

Antithrombin III↗

PubMed1991

Determination of the in vivo antigen-antibody affinity constant from the redistribution of desipramine in rats following administration of a desipramine-specific monoclonal antibody.

Quantitative expressions have been derived to determine the affinity constant for the in vivo interaction of an antigen with its elicited monoclonal antibody by analysing the redistribution of antigen following antibody administration. Using this method, the intrinsic binding constant for the interaction of subtoxic doses of DMI in rats with anti-TCA was found to be about two orders of magnitude less than the value obtained in vitro. The disparity is probably due to the presence of endogenous ligands for the antibody.

Animals↗

PubMed1991

Characterization of the immunochemical reactivity of fibrinogen fragments by competitive radioimmunoassay: an improved method of analysis.

Published results on the immunochemical reactivities of fibrinogen and fibrinogen fragments with fibrinogen-elicited antibodies that had been fractionated on the basis of preferential interaction with A alpha [Nagy, J. A., Meinwald, Y. C., and Scheraga, H. A. (1982), Biochemistry 21, 1794-1806] and B beta [Nagy, J. A., Meinwald, Y. C., and Scheraga, H. A. (1985) Biochemistry 24, 882-887] peptides of this bivalent antigen have been reinterpreted. First, the multivalent counterpart of the Scatchard analysis has been used to determine the intrinsic association constant for the interaction of antibody with [125I]fibrinogen, the radiolabeled ligand used in subsequent competitive binding studies. Second, the corresponding affinity constant for native fibrinogen has been evaluated from the relevant competitive radioimmunoassays by means of a quantitative analysis that takes into account the bivalency of both the radiolabeled and native fibrinogen molecules. Finally, affinity constants for the interactions of various fibrinogen fragments with antibody are also obtained by the procedure, and their magnitudes rationalized in terms of the equilibrium coexistence of unreactive (disordered) and native (functional) states of the fibrinogen peptides.

Binding, Competitive↗

PubMed1991

Experimental and theoretical studies of rate constant evaluation for the solute-matrix interaction in affinity chromatography.

In an investigation of the problem of determining kinetic parameters for the interaction of a solute with immobilized ligand sites on an affinity matrix, a combination of experimental studies and numerical simulations of frontal chromatography of methyl orange on Sephadex G-25 has yielded a simpler method than existing procedures for characterizing solute-matrix kinetics. A significant change in approach has entailed the direct evaluation of the kinetic contribution to boundary spreading from the flow-rate dependence of boundary variance under conditions of concentration-independent chromatographic migration (linear kinetics). This kinetic contribution is then interpreted in terms of an experimentally more appropriate form of a quantitative relationship for diffusion-free chromatographic migration (H. W. Hethcote and C. DeLisi, 1982, J. Chromatogr. 240, 269-281). Finally, the results of numerical simulations of concentration-dependent chromatographic migration (Langmuir kinetics) have indicated that rate constants should also be determinable under these conditions by extrapolation of their apparent values obtained by the above procedure to infinite dilution.

Alcohol Dehydrogenase↗

PubMed1991

Adaptation of the Müller method to allow quantitative characterization of the affinity and cross-reactivity of antibodies by competitive radioimmunoassay.

A quantitative expression is derived for the evaluation of antigen-antibody affinity constants from radioimmunoassays for the completely general situation in which antigen and antibody are both multivalent. The theoretical analysis is then extended to encompass quantitative characterization of the competitive inhibition observed in screening tests for cross-reactivity of antibody with structural analogs of the eliciting antigen. These procedures are illustrated with a radioimmunological study of the cross-reactivity of a desipramine-elicited monoclonal antibody with other tricyclic antidepressants. An unexpected finding to emerge from this immunochemical study is the demonstration that a single affinity constant suffices to describe the interaction of desipramine with a polyclonal antibody elicited by this univalent antigen.

Antibody Affinity↗

PubMed1991

Cimetidine: an inhibitor and an inducer of rat liver microsomal cytochrome P-450.

1. Cimetidine pretreatment of male Sprague-Dawley rats caused a significant increase in the specific content of total hepatic cytochrome P-450, supporting the hypothesis that this H2-receptor antagonist has monooxygenase induction effects. 2. Quantitative ultrastructural studies of liver of cimetidine-pretreated animals also supported this hypothesis in showing a significant proliferation of smooth endoplasmic reticulum. These ultrastructural changes were qualitatively similar to those produced by treatment of rats with phenobarbital, a well-characterized monooxygenase-inducing agent whose effects were studied for comparative purposes. 3. Competitive inhibition of metoprolol alpha-hydroxylation by cimetidine in liver microsomes prepared from untreated animals (Ki = 18.8 microM) was also demonstrated. 4. These results allowed testing of the hypothesis (Burnet et al. 1986) that inhibition of a defined monooxygenase should lead to induction of the synthesis of the relevant cytochrome P-450 isozyme. 5. The finding that metoprolol alpha-hydroxylase activity of liver microsomes was lowered, not elevated, by pretreatment of animals with cimetidine argues against the concept of a causal link between monooxygenase inhibition and induction.

Animals↗

PubMed1990

Thermodynamic nonideality as a probe of reversible protein unfolding effected by variations in pH and temperature: studies of ribonuclease.

Thermodynamic nonideality arising from the space-filling effect of added sucrose is employed to confirm that the reversible unfolding of ribonuclease A effected by acid may be described as an equilibrium between native and unfolded states of the enzyme. However, the extent of the volume change is far too small for the larger isomer to be the fully expanded state, a result signifying that the acid-mediated unfolding of ribonuclease does not conform with the two-state equilibrium model of protein denaturation. Although the thermal denaturation of ribonuclease A is characterized by a larger increase in volume, quantitative reappraisal of published results on the effects of glycerol on this transition at pH 2.8 (Gekko, K., and Timasheff, S. N., 1981 Biochemistry 20, 4677-4686) leads to an estimated volume increase that is much smaller than that inferred from hydrodynamic studies--a disparity attributed to the dual actions of glycerol as a space-filling solute and as a ligand that binds preferentially to the thermally unfolded form of the enzyme. Even in this unfavorable circumstance the fact that glycerol exerts a net excluded volume effect at least confirms that the thermal unfolding of ribonuclease A is an equilibrium transition between two discrete states. The strengths and limitations of using thermodynamic nonideality as a probe of the two-state equilibrium model of protein denaturation are discussed in the light of these findings.

Animals↗

PubMed1990

Interaction of cimetidine with human serum albumin.

Ultrafiltration studies have detected the existence of a weak interaction between cimetidine and human serum albumin, a finding supported by corresponding studies with this xenobiotic and bovine serum albumin. Furthermore, the binding characteristics of the interaction with human serum albumin (4 sites, K = 630 M-1) more than suffice to account for the proportion of protein-bound drug in the serum of patients subjected to cimetidine therapy. Thus, although alpha 1-acid glycoprotein is usually regarded as the specific transporter of basic drugs, the present evidence implicates albumin as the likely binding protein for cimetidine in serum.

Binding Sites↗

PubMed1990

Allowance for thermodynamic nonideality and Donnan effects in binding studies. Activity coefficients of charged ligands in the presence of albumin.

A combination of ultrafiltration with either equilibrium dialysis or frontal gel chromatography has been used to evaluate the effects of thermodynamic nonideality in mixtures of bovine serum albumin and charged ligands. Studies with methyl orange, chlorpromazine and chromate as ligand all demonstrated inadequacy of the Donnan effect for description of the difference between the concentrations of free ligand in a mixture and the protein-free phase with which it is in dialysis equilibrium. On the basis of a quantitative relationship derived for the situation in which Donnan and thermodynamic nonideality effects both operate, values of the second virial coefficient for albumin and ligand have been determined. For albumin and either methyl orange or chlorpromazine the magnitude of this second virial coefficient has been rationalized on the statistical-mechanical basis of excluded volume. For the albumin-chromate system, however, the thermodynamic nonideality was manifested as a negative deviation from Raoult's Law, in keeping with the classical behaviour of electrolyte ions. From the viewpoint of the characterization of ligand binding a unique feature of the ultrafiltration/gel chromatography and ultrafiltration/equilibrium dialysis methods is their ability to define not only the binding function but also the activity coefficient of ligand for a given acceptor-ligand mixture. Consequently, irrespective of whether the ligand is charged or uncharged, the intrinsic binding constant that is determined is the thermodynamic parameter instead of the apparent value that is obtained from methods based on assumed thermodynamic ideality.

Animals↗

PubMed1990

Preferential ligand binding to multi-state acceptor systems: the unexplored paradox of acceptor self-association that is ligand-mediated but detrimental to ligand binding.

Consideration is given to the interactions of ligand with self-associating acceptor systems for which preferential ligand binding is an ambiguous term, in that the acceptor species with greater affinity for ligand possesses relatively fewer binding sites. A paradoxical situation wherein ligand-mediated self-association is seemingly detrimental to ligand binding is shown to be the predicted outcome for a transient range of ligand concentrations. This outcome reflects the existence of a critical point in the dependence of the extent of acceptor self-association upon ligand concentration that coincides with a cross-over point of ligand-binding curves for different, fixed total concentrations of acceptor. By classical differentiation methods the conditions for the existence of these critical points are established not only for two-state acceptor systems but also for three-state acceptor systems in which the ligand-binding form of monomer also undergoes reversible isomerization to an inactive state. Similar procedures are used to comment upon the forms of binding curves for the three-state acceptor systems, the Scatchard representations of which may exhibit as many as three critical points (two maxima and a minimum). This delineation of quantitative expressions for critical points and other distinctive features associated with the conflicting interplay of ligand-binding and self-association behaviour should provide a more definitive means of characterizing systems with one acceptor state the preferred binding form on affinity grounds but with the other the preferred state from the viewpoint of binding-site numbers.

Animals↗

PubMed1990

Thermodynamic nonideality in macromolecular solutions. Evaluation of parameters for the prediction of covolume effects.

Second virial coefficients and hence covolumes for self-interaction of five proteins, viz. ribonuclease, ovalbumin, bovine serum albumin, catalase and alpha-crystallin, have been determined by analyzing the concentration dependence of the partition coefficient obtained from frontal chromatographic studies on either Fractogel TSK HW55 or porous glass beads. The resulting estimates of the effective radii essentially duplicate their Stokes counterparts and thereby provide further justification for assuming the approximate identity of the thermodynamic and hydrodynamic radii of hydrated globular proteins. Gel chromatographic evaluation of second virial coefficients for protein/dextran systems has led to elimination of the sphere/sphere model as a valid thermodynamic description of the space-filling effects in protein/polymer mixtures, since it does not predict the observed independence of covolume, expressed per unit mass of polymer, upon size of the polymer. This requirement is met by the sphere/rod model [Edmond, E. & Ogston, A. G. (1968) Biochem. J. 109, 569-576] and also by the sphere/flexible-segment model [Hermans, J. (1982) J. Chem. Phys. 77, 2193-2203]. Furthermore, similar studies of the effect of solute radius on covolume for interaction with dextran T70 attest to the adequacy of either model for predicting the thermodynamic nonideality arising from the inclusion of dextrans in protein solutions, and also provide the relevant calibration of the model.

Catalase↗

PubMed1990

Interactions of glycolytic enzymes with erythrocyte membranes.

Partition equilibrium experiments have been used to characterize the interactions of erythrocyte ghosts with four glycolytic enzymes, namely aldolase, glyceraldehyde-3-phosphate dehydrogenase, phosphofructokinase and lactate dehydrogenase, in 5 mM sodium phosphate buffer (pH 7.4). For each of these tetrameric enzymes a single intrinsic association constant sufficed to describe its interaction with erythrocyte matrix sites, the membrane capacity for the first three enzymes coinciding with the band 3 protein content. For lactate dehydrogenase the erythrocyte membrane capacity was twice as great. The membrane interactions of aldolase and glyceraldehyde-3-phosphate dehydrogenase were mutually inhibitory, as were those involving either of these enzymes and lactate dehydrogenase. Although the binding of phosphofructokinase to erythrocyte membranes was inhibited by aldolase, there was a transient concentration range of aldolase for which its interaction with matrix sites was enhanced by the presence of phosphofructokinase. In the presence of a moderate concentration of bovine serum albumin (15 mg/ml) the binding of aldolase to erythrocyte ghosts was enhanced in accordance with the prediction of thermodynamic nonideality based on excluded volume. At higher concentrations of albumin, however, the measured association constant decreased due to very weak binding of the space-filling protein to either the enzyme or the erythrocyte membrane. The implications of these findings are discussed in relation to the likely subcellular distribution of glycolytic enzymes in the red blood cell.

Animals↗

PubMed1990

Effect of calcium ion on the dimerization of alpha-chymotrypsin.

Results of a sedimentation equilibrium study of the inhibitory effect of calcium ion on the dimerization of alpha-chymotrypsin (pH 3.9, I 0.2) are used to establish that the phenomenon does not reflect increased electrostatic repulsion between Ca2(+)-saturated enzyme molecules, but rather the displacement of the monomer-dimer equilibrium by the specific, weak interaction of metal ion with a single site on monomeric enzyme.

Calcium↗

PubMed1990

Evidence of neuropeptide bivalency in the interaction of substance P with cerebroside sulfate.

A precipitin effect has been observed with mixtures of cerebroside sulfate and the neuropeptide substnace P. This phenomenon is attributed to multivalency of the lipid due to its existence in micellar form, and to bivalency of substance P. One of those neuropeptide sites is almost certainly the basic residue(s) located at the N-terminal of substance P, whereas the hydrophobic residues at the C-terminus are suggested as candidates for the other site on the basis of turbidimetric, circular dichroic, and fluorometric studies. An intrinsic association constant of 3.6 x 10(4)M-1 has been obtained from the cerebroside sulfate concentration associated with maximal turbidity of mixtures containing a fixed concentration of the neuropeptide.

Binding Sites↗

PubMed1989

Equilibrium partition studies of the myofibrillar interactions of glycolytic enzymes.

The interactions of several glycolytic enzymes with muscle myofibrils in imidazole-chloride buffer (pH 6.8, I 0.158) have been investigated by equilibrium partition studies. Results for aldolase, glyceraldehyde-3-phosphate dehydrogenase, lactate dehydrogenase, and phosphofructokinase are interpreted in terms of a myofibrillar capacity of 76 nmol/g protein and a single intrinsic association constant for each tetravalent enzyme with matrix sites. The existence of separate myofibrillar sites for aldolase and glyceraldehyde-3-phosphate dehydrogenase is established by demonstrating independence of the binding of each enzyme upon the presence of the other. Although this investigation provides further physicochemical support for myofibrillar adsorption of glycolytic enzymes in the cellular environment, its findings are incompatible with the proposition (B. I. Kurganov, N. P. Sugrobova, and L. S. Mil'man (1985) J. Theor. Biol. 116, 509-526) that the phenomenon reflects the formation of a specific multienzyme complex attached to the myofibril.

Animals↗