三齿草藤(Vicia bungei Ohwi)凝集素的细胞表面受体研究

应用亲和层析法从三齿草藤(Vicia bungei Ohwi)种子中纯化的三齿草藤凝集素(VBL),可以凝集兔和豚鼠的红细胞,也可凝集人、牛和羊的精细胞,说明这些细胞表面存在有VBL的受体。用FITC和~(125)I进行标记,可得到FITC-VBL和~(125)I-VBL,其生物学活性不受影响。氯胺T法的标记率可达55％;应用FITC-VBL研究牛精细胞和兔红细胞膜上VBL受体的分布,发现二者由胞膜上受体分布据不一致。VBL与牛精细胞结合条件的正交试验表明细胞浓度的影响最大。用不同量的未标记的VBL对~(125)I-VBL与兔红细胞和人精细胞的结合实验,以Scatchard法作图,兔红细胞得一类似于双曲线的凹形曲线,提示该细胞膜上受体的性质有所不同,而人精细胞却有很大差异。若以兔红细胞膜上存在有高低亲和力两种受体进行计算,可求得结合常数和每个细胞上的受体数。应用几种单糖和外源凝集素影响~(125)I-VBL与兔红细胞的结合,当单糖(D-Man,D-Glc)浓度为0.01M时,相对结合率开始急剧下降,单糖浓度若增至0.1M时,其相对结合率仅为40％,而PHA-P和SML浓度为1mg/ml时,相对结合率开始下降,当浓度达10mg/ml时,相对结合率下降至30％左右。     

Misuse of nonlinear Scatchard plots

Scatchard plots--plots of bound/free ligand vs bound ligand--are a common graphical presentation of binding data. They are often nonlinear. Despite examples of correct usage and several articles calling attention to incorrect treatment of Scatchard plots, erroneous interpretations of nonlinear Scatchard plots remain frequent; plots are resolved incorrectly into two or more linear components which have no relation to an acceptable binding model. Correct analysis requires determination, usually by computer, of numerical values of the binding parameters that give the best nonlinear fit to an appropriate model, examples of which are specified.     

Binding of hormones to receptors. An alternative explanation of nonlinear Scatchard plots.

Binding of 1251-labeled hormones to receptors can usually be inhibited by addition of unlabeled hormone. In analyzing such binding-inhibition data, it is commonly assumed that both labeled and unlabeled hormones are bound with equal affinity. When this assumption is made incorrectly, an artifactually nonlinear Scatchard plot results. Equations to describe these nonlinear Scatchard plots are derived. These results are discussed with regard to previously published observations of nonlinear Scatchard plots for binding of insulin to its receptor.     

Interactions between micellar ligand systems and acceptors: Forms of binding curves

A previously formulated expression describing the competitive binding to an acceptor of two states of a ligand, monomeric and polymeric, coexisting in equilibrium is examined in terms of the different forms of Scatchard plots which may arise in cases of exclusive and of preferential binding of the ligand states. It is shown by differentiation of the binding equation written in Scatchard format, and by numerical examples, that exclusive binding of the monomeric form of ligand leads to Scatchard plots that are either sigmoidal or convex to the abscissa, whereas exclusive binding of the polymeric form results in plots concave to the abscissa and exhibiting a maximum. Particular attention is given to Scatchard plots which possess two critical points, a situation which is shown to be possible when the polymeric form of ligand binds preferentially (but not exclusively) to the acceptor. The two-state ligand concept is especially pertinent to solutes capable of globular micelle formation and several examples are cited of binding studies which have been conducted with such micellar systems. Of these, the chlorpromazine-brain tubulin system is given detailed consideration in order to illustrate the use of the present theory in describing the binding results which exhibit two critical points when plotted in Scatchard format.     

Conditions for the existence of critical points in scatchard plots of binding results

An equation is presented in Scatchard format which describes the binding of a ligand to a two-state acceptor system (either isomerizing or polymerizing). It is used to formulate a general set of conditions for the existence of critical points in Scatchard plots and this set is explored in detail for particular cases. Explicit relations between the thermodynamic parameters governing the binding are thereby obtained which permit discussion of the boundaries of domains within which critical points may exist. Moreover, several items of evidence are presented which show that there is an exact correspondence between the appearance of critical points in Scatchard plots and points of inflexion in associated binding curves examples are presented where zero, one or more critical points and points of inflexion are observed. Finally, the effects on preferential binding of the variation of the environmental parameters, temperature and pressure (and for acceptor polymerization, the total acceptor concentration) are discussed in terms of the derived conditions of existence of critical points in Scatchard plots and their equivalent domains of sigmoidality of binding curves.     

Simulation of Association Curves and ‘Scatchard’ Plots of Binding Reactions Where Ligand and Receptor are Degraded or Internalized

Abstract

Frequently during the course of binding to a receptor, ligand is degraded. In some preparations receptor is degraded. And with isolated cell preparations, ligand and/or receptor may be internalized. Here we present a mathematical model in which the binding and other reactions are combined. The resulting set of differential equations is solved numerically to simulate association curves and the resulting values of bound and free ligand are used to construct Scatchard plots. Where non-ideal conditions exist, the Scatchard plots are generally curvilinear. Dependence of this curvilinearity - on time of measurement of free and bound ligand, on degradation and internalization of ligand, and on degradation and internalization of receptor - is shown. Equilibrium constants derived from the Scatchard plots are generally incorrect but the derived receptor concentration is often correct. The simulations suggest experimental possibilities for distinction among the several side reactions in ligand-receptor binding systems.     

Purification of DT-diaphorase by affinity chromatography. Occurrence of two subunits and nonlinear Dixon and Scatchard plots of the inhibition by anticoagulants.

A new purification procedure based on affinity chromatography of rat liver DT-diaphorase on Sepharose-bound dicoumarol is described. The native enzyme has a molecular weight of 55,000 and contains 1 mol of flavin-adenine dinucleotide per mol of enzyme, in agreement with earlier reports. Sodium dodecyl electrophoresis sulfate-gel reveals the occurrence of two subunits of about equal molecular weight (27,000). Dixon plots of the inhibition of the native enzyme by either dicoumarol or 2-pivaloyl-1,3-indandione are nonlinear. Scatchard plots of dicoumarol binding in the absence or presence of other anticoagulants, (Warfarin and 2-pivaloyl-1,3-indandione) are also nonlinear. The nonlinear Dixon and Scatchard plots are interpreted as reflecting the existence of more than one inhibitor-binding site. The kinetic data are discussed in relation to the subunit structure of the enzyme.     

Robust regression-based analysis of drug-nucleic acid binding

Outlier detection can be very important in analyzing data from Scatchard plots. In this study, a robust (outlier-resistant) regression procedure was used in conjunction with a Scatchard plot to study the binding of the methylphenazinium cation with double-stranded DNA. The procedures, their results, and their advantages are discussed.     

A critical interpretation of experiments of binding of peptide hormone to specific receptors by computer modelling

Many investigations dealing with the interaction of peptide hormones and specific cell membrane receptors imply the existence of two classes of independent binding sites. One class is characterized by high affinity and low capacity, the other one by low affinity and high capacity. This conclusion has been derived from the fact that the Scatchard plots of binding data show a significant upward curvature. Using a more precise and critical method of evaluation these findings probably must be revised in some cases. Other conclusions taken from linear plots concerning the regulation of hormone receptors should be discussed more carefully. Some sources of errors caused by an uncritical interpretation of Scatchard plots are demonstrated.     

The Ca2+ binding to deionized monomerized and to retinal removed bacteriorhodopsin.

In our continuing effort to characterize the metal cation binding in bacteriorhodopsin (bR) using Ca(2+)-specific electrodes, potentiometric titration was carried out on deionized solubilized bR (containing monomeric units) and deionized bacterioopsin (bR with its retinal removed). Scatchard plots were analyzed. The monomer was found to have plots similar to those of the trimer, suggesting that the binding sites in bR are localized within the protein monomer unit and not between the molecules within the trimer structure. This also supports the previous assumption that the curvature in the Scatchard plot of regenerated bR is not due to cooperativity of metal cation within the trimer, but rather due to multiple sites. Recent studies further support the finding that the curved Scatchard plot is not due to the cooperativity between the metal ions in the two high affinity sites, wherever they are. The results of the analysis of the Scatchard plot for deionized bacterioopsin have shown a change in the binding characteristics of the high affinity but not the low affinity sites from that observed in bR. This result supports previous conclusions that metal cations in the high affinity sites are not far from the retinal cavity.     

Implications of epidermal growth factor (EGF) induced egf receptor aggregation.

To investigate the role of receptor aggregation in EGF binding, we construct a mathematical model describing receptor dimerization (and higher levels of aggregation) that permits an analysis of the influence of receptor aggregation on ligand binding. We answer two questions: (a) Can Scatchard plots of EGF binding data be analyzed productively in terms of two noninteracting receptor populations with different affinities if EGF induced receptor aggregation occurs? No. If two affinities characterize aggregated and monomeric EGF receptors, we show that the Scatchard plot should have curvature characteristic of positively cooperative binding, the opposite of that observed. Thus, the interpretation that the high affinity population represents aggregated receptors and the low affinity population nonaggregated receptors is wrong. If the two populations are interpreted without reference to receptor aggregation, an important determinant of Scatchard plot shape is ignored. (b) Can a model for EGF receptor aggregation and EGF binding be consistent with the "negative curvature" (i.e., curvature characteristic of negatively cooperative binding) observed in most Scatchard plots of EGF binding data? Yes. In addition, the restrictions on the model parameters required to obtain negatively curved Scatchard plots provide new information about binding and aggregation. In particular, EGF binding to aggregated receptors must be negatively cooperative, i.e., binding to a receptor in a dimer (or higher oligomer) having one receptor already bound occurs with lower affinity than the initial binding event. A third question we consider is whether the model we present can be used to detect the presence of mechanisms other than receptor aggregation that are contributing to Scatchard plot curvature. For the membrane and cell binding data we analyzed, the best least squares fits of the model to each of the four data sets deviate systematically from the data, indicating that additional factors are also important in shaping the binding curves. Because we have controlled experimentally for many sources of receptor heterogeneity, we have limited the potential explanations for residual Scatchard plot curvature.     

Simulation of Association Curves and ‘Scatchard’ Plots of Binding Reactions where Ligand and Receptor are Degraded or Internalized

Abstract

Frequently during the course of binding to a receptor, ligand is degraded. In some preparations receptor is degraded. And with isolated cell preparations, ligand and/or receptor are internalized. Here we present a mathematical model for the combined binding and other reactions which gives useful information about the behaviour of such systems. The set of differential equations is solved numerically to simulate association curves and the resulting values of bound and free ligand are used to construct Scatchard plots. Where non-ideal conditions exist, the Scatchard plots are generally curvilinear. Dependence of this curvilinearity on time of measurement of free and bound ligand, on degradation and internalization of ligand and on degradation and internalization of receptor is shown. Equilibrium constants derived from the Scatchard plots are generally incorrect but the derived receptor concentration is often correct. The simulations lead to possibilities for distinction among the several side reactions in ligand-receptor binding systems.     

Unusual behaviour of the interaction of sodium n-dodecyl sulphate with calf thymus histone H2B in aqueous solution

The binding of sodium n-dodecyl sulphate (SDS) to calf thymus histone H2B was studied in the pH range 3.2-10 by equilibrium dialysis at 27 and 37 degrees C. The binding data have been used in terms of the Scatchard equation showing unusual plots with minima. No theoretical model gives Scatchard plots with such conditions, except for a combination of two types of binding with large differences in the Hill coefficients and binding affinity, i.e. a combination of negatively and positively cooperative binding sites.     

Effect of the incomplete separation of bound and free ligand on binding measurements

An incomplete separation of free and acceptor-bound ligand causes underestimation of specific binding even though the determinations are corrected with blank or nonspecific binding values. If the failure of the experimental procedure causes contamination of bound ligand with free ligand, Scatchard plots are linear, though their slopes and abscissa intercepts are different from the true ones. On the other hand, if the ligand-acceptor complex is incompletely recovered, Scatchard plots are curvilinear with downward concavity. These problems can be overcome if the separable fractions of free and bound ligand are measured and suitable corrections are applied.The separation of free and receptor-bound ¹²⁵I-labeled human growth hormone by a precipitation method is taken as an example of the procedure.     

Binding of mithramycin to DNA in the presence of second drugs

Comparative DNA equilibrium binding studies with mithramycin (MTR) and ethidium bromide in the presence and in the absence of second drugs were investigated by spectral titrations. Unusual curvatures (in contrast to those due to neighbor exclusion or anticooperativity) are found in the Scatchard plots of MTR-DNA titrations in the presence of netropsin, a minor-groove binder. Parallel studies with ethidium bromide indicate that although the presence of netropsin significantly reduces the binding ability of ethidium, no unusually curved Scatchard plots are obtained. The unusual curvature exhibited by the Scatchard plots of MTR titrations in the presence of netropsin indicates that the binding of netropsin greatly affects the MTR binding to DNA and can be simulated by an explicit incorporation of the second drug-DNA interaction in the binding formalism. Since netropsin is a minor-groove binder, its interference with the binding of MTR is in accord with the notion that MTR also binds at this groove. The observation of negligible effects on the DNA binding ability of MTR in the presence of either a major-groove or a phosphate group binder lends further support to this conclusion. Consistent with its guanine specificity, studies with synthetic polynucleotides suggest that MTR exhibits negligible affinity for poly(dA-dT).poly(dA-dT) or poly(dA).poly(dT).(ABSTRACT TRUNCATED AT 250 WORDS)     

A note on the quantitative properties of McGhee-von Hippel model

Exact closed-form expressions are presented for the properties of the McGhee-von Hippel model of non-specific binding of large ligands to one-dimensional homogeneous lattices. These properties include the midpoint location and the slope at the middle point of the binding isotherms (v varies with ln L plots), the location and magnitude of the maximum, as well as the location of the inflection point, in the Scatchard plots (v/L varies with v plots).     

Methods for the estimation of receptor capacity of cyclic AMP-dependent protein kinases.

The dissociation of cyclic AMP-dependent protein kinase to give two catalytic subunits results in data for the binding reaction that cannot be interpreted by the method of Scatchard. Linear plots that allow determination of total receptor capacity and equilibrium association constant are described.     

Langmuir and scatchard parameters do not describe the binding of Actinomyces viscosus to saliva-treated hydroxyapatite

The binding of Actinomyces viscosus T14V to saliva-treated spheroidal hydroxyapatite (SHA) beads was studied. The association constant (K) and the total number of binding sites (N) obtained from the Langmuir plots were in good agreement with those reported by other workers (approx. 3 X 10(-8) and 3 X 10(8), respectively). The values for N obtained from Scatchard plots differed from those obtained from Langmuir plots by factors of 10(6) or more. These results suggest that either these equations are inappropriate to describe binding or certain assumptions regarding this system are not being met. The use of these models requires, among other constraints, that the process be reversible and that measurements be taken at equilibrium. A method was developed which allowed a close examination of the equilibrium dynamics without perturbation of the system. The results suggest that the adsorption process is only poorly reversible. Adsorption to SHA was not at equilibrium after 1.5 h. Even when bacteria were allowed to adsorb for longer periods, and the system appeared to approach equilibrium, the increased time of adherence did not significantly alter the derived K or N values. Our results suggest that the use of Scatchard and Langmuir plots is inappropriate to describe binding of A. viscosus to SHA.     

An extended Scatchard analysis for competitively bound ligands and its application to cyclic adenosine-3',5'-monophosphate and cyclic 5'-amido-5'-deoxyadenosine-3', 5'-monophosphate binding to beef skeletal muscle protein.

A method for determining the dissociation constants of ligands and ligand analogs is described. It is based on competition binding studies in the presence of an isotope-labeled, or otherwise measurable, ligand and suitable analog concentrations.The steps used are determination of (1) the maximal amount of radioactive ligand that can be bound, (2) the slopes and intercepts from Scatchard plots at different analog concentrations and (3) the values for the dissociation constants of radioactive ligand and ligand analog from replots of the reciprocals of the slopes and intercepts obtained from the Scatchard plots. Application of the method to a cyclic AMP-binding protein from beef muscle is demonstrated, yielding dissociation constants of 2.10-9 M for cyclic (3H) AMP and cyclic AMP, and 3.10-5 M for cyclic 5'-amido-5'-deoxyadenosine-3', 5'-monophosphate.     

Estradiol- and estriol-binding in human benign prostatic hyperplasia.

Binding of the natural estrogens, estradiol and estriol, was investigated, in 34 samples of human benign prostatic hypertrophy (BPH) tissue, using Scatchard analysis and agar gel electrophoresis. Saturation binding analysis using a wide range of concentrations of both ligands resulted in curvilinear Scatchard plots. This confirmed the presence of two binding forms for estradiol: a true estrogen receptor, and a protein with lower affinity and higher capacity. Both binding species were also demonstrated and quantified with estriol. The electrophoretic process, after incubation at low and high ligand concentrations also resulted in separation, for both estrogens, of two binding peaks. They are probably two distinct forms of the low affinity, high capacity binding measured by Scatchard. The procedure used in our laboratory was not able to provide accurate determination of the concentrations of these binding forms. Possible modifications to alleviate these drawbacks are discussed.