Ca<sup>2+</sup>-binding activity of protein isolated from sarcotubular membranes

Ha, Bertrand; Ej, Masoro; Ohnishi, Tsuyoshi; Bp, Yu

doi:10.1021/bi00796a007

Cited by 24 publications

(4 citation statements)

References 20 publications

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“…Graphical solutions, based on the properties of asymptotes to the binding curves [14] or the representation of Rosenthal, have been used. The latter technique can be applied to a semiautomatic computer program, which performs the iterations requested [15]. A mathematical non-graphical resolution has been proposed by Hart [16].…”

Section: Two Cu Fegories Of Specijic Binding Sitesmentioning

confidence: 99%

Determination of Protein‐Ligand Binding Constants at Equilibrium in Biological Samples

Blondeau¹,

Röbel²

1975

European Journal of Biochemistry

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Protein-ligand complexes can be separated functionally into two classes. "Specific" binding is characterized, in relative terms, by a high affinity for the ligand and a low binding capacity. "Nonspecific" binding is characterized by a low affinity and a very large capacity. The calculation of equilibrium binding constants for any specific protein-ligand interaction requires the exact determination of the unbound ligand concentration and the specifically bound ligand concentration. These determinations usually require corrections for the contribution of non-specific binding. The use of two correction terms, kn andf, is proposed: kn is the product of the affinity constant k times the number of binding sites M of the non-specific components, whilef is the fraction of the non-specific binding included in the experimental estimates of bound ligand. Several theoretical solutions using these terms are proposed for the calculation of specific binding constants. The practical choice of the correction factor may be different when the simultaneous measurement of the affinity constant and maximum number of binding sites, or when only the latter, is desired. In the case of complex binding systems containing more than one specific component, the individual constants can be determined by non-graphical methods, using computer-aided iterative statistical calculations. A complete solution is given for a system containing two specific plus non-specific interactions and actual experiments are reported for steroid hormone-receptor complexes.The present work deals with the theoretical aspects of the determination of specific binding constants at equilibrium in complex binding systems. In most biological samples, any given ligand interacts with several binding components. These components can be separated functionally into two classes. 'Specific' Symbols and Abhreviations. T = total concentration of radioactive ligand incubated with the biological extract (moles/litre) ; U = concentration of free ligand at equilibrium (moles/litre); Bs = concentration of ligand bound to high-affinity, low-capacity macromolecules, at equilibrium (moles/litre); EN, = concentration of ligand bound to macromolecules of low affinity and practically unlimited capacity, at equilibrium (moles/litre) ; f = fraction of the non-specific binding (Bhs) included in the measurement of B ; kn = affinity constant x number of binding sites of the non-specific binding component(s) ; (in the experimental conditions used. the individual non-specific binding components are not discriminated and only the sum of the kn values of these non-specific binding components can be measured); K j = association constant of the .jth category of specific binding sites (litre/mole); N j = total number of binding sites of thejth category of specific binding sites (moles/litre).

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Section: Two Cu Fegories Of Specijic Binding Sitesmentioning

confidence: 99%

Determination of Protein‐Ligand Binding Constants at Equilibrium in Biological Samples

Blondeau¹,

Röbel²

1975

European Journal of Biochemistry

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“…Least-squares parameter fitting was also used by Nagy [lo]. A graphic parameter fitting procedure has been described by Berson and Yalow [ll], and Rosenthal [12], while others [13,14] used a semiautomated parameter fitting on a small computer. The aim of the present investigation was to study the determination of the parameters of binding systems with different means of expenditure.…”

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confidence: 99%

Determination of Binding Parameters from Scatchard Plots

Weder

Schildknecht

Lutz

et al. 1974

European Journal of Biochemistry

146

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Procedures for graphic and numerical parameter fitting from Scatchard plots for the case of two different independent sets of equivalent binding sites are described. These methods are compared with other procedures and their advantages and disadvantages are discussed. Estimates of how much one parameter can be changed without changing the Scatchard curve significantly are made. This variability of the parameters is discussed. It is shown that low saturation points are of considerable importance in evaluating the binding parameters of the high affinity binding site. An example of the parameter fitting procedures is given and the utility and limitations of the procedures are discussed. It has been stated [6] that a logarithmic plot should be used to exclude other forms of data representation. The logarithmic scale allows one t o plot all theoretically obtainable data. The doublereciprocaI plot [7] and the Scott or half-reciprocal plot [8] have open upper limits on the abscissa as has the absorption isotherm. I n the Scatchard plot, all theoretically obtainable data can be plotted provided that the degree of binding is used on the abscissa and the nonspecific binding sites with large binding capacity are absent. An objection to the Sctachard plot was that linearisation of binding data, which is only possible in the rare case of only one single set of binding sites, should result in a loss of information [4]. Nevertheless, we studied the representation of binding data with the Scatchard plot, mainly because of its wide-spead use, but being all the time aware of its drawbacks in certain cases.A similar study has been undertaken by Feldman 191, where a least-squares parameter fitting has been used for the numerical method. Least-squares parameter fitting was also used by Nagy [lo]. A graphic parameter fitting procedure has been described by Berson and Yalow [ll], and Rosenthal [12], while others [13,14] used a semiautomated parameter fitting on a small computer. The aim of the present investigation was to study the determination of the parameters of binding systems with different means of expenditure. The graphical and numerical curve fitting have been studied using a programmable calculator and a computer. The numerical parameter fitting of the computer program does not use least-squares regression, therefore some difficulties could be overcome. To test the applicability of the fitting procedure a practical example will be given as an illustration. BASIC RELATIONSHIPSThe outlined procedures are based on the following assumptions : all ligands are identical, the activity (chemical, biological) will not be altered except by binding, two independent sets of binding sites are present, all sites of each set are equivalent (identical), there is no cooperativity, each set of binding sites obeys the mass law action.At equilibrium for each system containing one single set of binding sites, the Scatchard plot [3] gives a straight line following the equation Eur. J. Biochem. 42 (1974)

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“…It is quite possible that two (or more than two) subunit molecules are cooperatively involved in the Ca++ binding by sarcoplasmic reticulum. Further studies on the Ca++ binding of Fr-2 have been reported elsewhere (13). Open circles are experimental data.…”

Section: Discussionmentioning

confidence: 97%

Analysis of a Calcium Ion Binding System Composed of Two Different Sites

Ohnishi¹,

Masoro²,

Bertrand³

et al. 1972

Biophysical Journal

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Using murexide (Mx), a metallochromic indicator, and a dual wavelength spectrophotometer with a high signal-to-noise ratio, the Ca(++) binding in a system containing two classes of binding sites was studied. Solutions with solute containing one or two classes of Ca(++) binding sites and without such solute were titrated with Ca(++) using Mx as an indicator of free Ca(++) concentration. Since curvilinear Scatchard plots are obtained from titration curves of solutes containing two classes of binding sites, a computer program was developed to resolve such plots into two linear partial plots, each corresponding to a single class of binding site. The validity of the procedure was examined with solutions of ethylene glycol bis(beta-aminoethyl)-N-N'-tetraacetic acid, adenosine triphosphate (EGTA, ATP), or a mixture thereof. The method was also applied to biological material and it was found that a protein fraction isolated from rat skeletal muscle sarcotubular membranes, termed Fraction-2 (Fr-2), has two classes of binding sites for Ca(++); the association constants of the high affinity site and low affinity site are 4.3 x 10(5) M(-1) and 9 x 10(3) M(-1), respectively. The advantages and limitations of this methodology are discussed.

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Ca²⁺-binding activity of protein isolated from sarcotubular membranes

Cited by 24 publications

References 20 publications

Determination of Protein‐Ligand Binding Constants at Equilibrium in Biological Samples

Determination of Protein‐Ligand Binding Constants at Equilibrium in Biological Samples

Determination of Binding Parameters from Scatchard Plots

Analysis of a Calcium Ion Binding System Composed of Two Different Sites

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Ca2+-binding activity of protein isolated from sarcotubular membranes

Cited by 24 publications

References 20 publications

Determination of Protein‐Ligand Binding Constants at Equilibrium in Biological Samples

Determination of Protein‐Ligand Binding Constants at Equilibrium in Biological Samples

Determination of Binding Parameters from Scatchard Plots

Analysis of a Calcium Ion Binding System Composed of Two Different Sites

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Ca²⁺-binding activity of protein isolated from sarcotubular membranes