Showing papers on "Cooperative binding published in 1979"

Equilibrium binding of [3H]tubocurarine and [3H]acetylcholine by Torpedo postsynaptic membranes: stoichiometry and ligand interactions.

Abstract: Studies are presented of the equilibrium binding of [3H]-d-tubocurarine (dTC) and [3H]acetylcholine (AcCh) to Torpedo postsynaptic membranes. The saturable binding of [3H]dTC is characterized by two affinities: Kd1 = 33 +/- 6 nM and Kd2 = 7.7 +/- 4.6 microM, with equal numbers of binding sites. Both components are completely inhibited by pretreatment with excess alpha-bungarotoxin or 100 microM nonradioactive dTC and competitively inhibited by carbamylcholine with a KI = 100 nM, but not affected by the local anesthetics dimethisoquin, proadifen, and meproadifen. The biphasic nature of [3H]dTC binding was unaltered in solutions of low ionic strength and by preparation of Torpedo membranes in the presence of N-ethylmaleimide, a treatment which yields dimeric AcCJ receptors. dTC competitively inhibits the binding of [3H]AcCH and decreases the fluorescence of 1-(5-dimethylaminonaphthalene-1-sulfonamido)ethane-2-trimethylammonium (Dns-Chol) in a manner quantitatively consistent with its directly measured binding properties. It decreases the initial rate of 3H-labeled Naja nigricollis alpha-toxin binding by 50% at 60 nM with an apparent Hill coefficient of 0.58. The stoichiometry of total dTC, AcCh, and alpha-neurotoxin binding sites in Torpedo membranes was determined by radiochemical techniques and by a novel fluorescence assay utilizing Dns-Chol as an indicator, yielding ratios of 0.9 +/- 0.1:0.9 +/- 0.2:1, respectively. The biphasic equilibrium binding function is not unique to dTC since other ligands inhibited [3h]acCh binding in a biphasic manner with apparent inhibition constants as follows: gallamine triethiodide (K11 = 2 microM, K12 = 1 mM); Me2dTC (K11 = 500 nM, K12 = 10 microM); decamethonium (K11 = 100 nM, K12 = 1.6 microM). Carbamylcholine, however, inhibited [3H]AcCh binding with a single KI = 100 nM. The observed competition between those ligands and [3H] AcCh cannot be completely accounted for by competitive interaction with two different affinities, and the deviations are discussed in terms of the positive cooperativity of the [3H] AcCh binding function itself. It is concluded that dTC binds only to the AcCh sites in Torpedo membranes and that those sites display two affinities for dTC but only one for AcCh.

Analysis of cooperativity and ion effects in the interaction of quinacrine with DNA.

Abstract: The interaction of quinacrine with calf thymus DNA was monitored at several different ionic strengths using spectrophotometric and equilibrium dialysis techniques. The binding results can be explained, assuming each base pair is a potential binding site, using a model containing two negative cooperative effects: (1) ligand exclusion at binding sites adjacent to a filled binding site and (2) ligand–ligand negative cooperativity at adjacent filled binding sites. The logarithm of the observed equilibrium constant (Kobs) determined by this model varies linearily with log[Na+], as predicted by the ion condensation theory for polyelectrolytes. When the log Kobs plot is correlated for sodium release by DNA in the intercalation conformational change, the predicted number of ion pairs between the ligand and DNA is approximately two, as expected for the quinacrine dication. Even though Kobs depends strongly on ionic strength, the ligand negative cooperativity parameter ω was found to be indpendent of ionic strength within experimental error. This finding is also in agreement with the ion condensation theory, which predicts a relatively constant amount of condensed counterion on the DNA double helix over this ionic strength range. Drugs would, therefore, experience a relatively constant ionic environment when complexed to DNA even though the ionic conditions of the solvent could change considerably.

Alpha noradrenergic receptors in brain membranes: sodium, magnesium and guanyl nucleotides modulate agonist binding.

Abstract: Binding of [3H]clonidine to alpha noradrenergic receptors in rat brain is inhibited by monovalent cations (Na+>Li+>K+), stimulated by magnesium ion and inhibited by guanyl nucleotides In the presence of 1 mM EDTA the receptors bind tritiated clonidine in a noncooperative fashion at a single site with a K a(association constant) of 012 nM−1 In the presence of magnesium the affinity of the receptors increases by a factor of two (K a=023 nM−1) The increase of affinity is attributed to a two-fold decrease in the dissociation rate constant In the presence of sodium ions the concentration of binding sites is not changed but Scatchard plots are now curvilinear indicating either heterogeneity of the receptors or negative cooperativity in ligand binding This effect of sodium ions is not influenced by the presence of magnesium The conversion into the sodium-liganded state is rapid; it is complete within 60 s at 30° C The effects of the guanyl nucleotides on clonidine binding are complex: In the presence saturating concentrations of sodium ions they cannot inhibit clonidine binding except when free magnesium (>1 mM) is present Without added sodium and in the presence of 1 mM EDTA the rank order of potencies is: GDP≧GTP>Gpp(NH)p In the presence of 10 mM magnesium the rank order is reversed: Gpp(NH)p ≫ GTP≧GDP The apparent affinity of the nucleotides for inhibition of clonidine binding is also changed by magnesium The affinity of Gpp(NH)p increases about 100-fold by addition of magnesium ion

Studies of reversible and irreversible interactions of an alkylating agonist with Torpedo californica acetylcholine receptor in membrane-bound and purified states.

Abstract: The interaction of a cholinergic depolarizing agent, bromoacetylcholine, with acetylcholine receptor (AcChR) enriched membrane fragments and Triton-solubilized, purified AcChR from Torpedo californica has been studied. The reagent bound to membrane-bound AcChR reversibly with an apparent dissociation constant of 16 +/- 1 nM at equilibrium. This 600-fold higher affinity for the receptor than found from physiological studies [Kact congruent to 10 micrometers; Karlin, A. (1973) Fed. Proc. Fed. Am. Soc. Exp. Biol. 32, 1847--1853] can be attributed to a ligand-induced affinity change of the membrane-bound receptor upon preincubation with bromoacetylcholine. At equilibrium [3H]bromoacetylcholine, like acetylcholine, bound to half the number of alpha-bungarotoxin sites present in the preparation without apparent positive cooperativity, and this binding was competitively inhibited by acetylcholine. In the presence of dithiothreitol, [3H]bromoacetylcholine irreversibly alkylated both membrane-bound and solubilized, purified acetylcholine receptor, with a stoichiometry identical with that for reversible binding. NaDodSO4-polyacrylamide gel electrophoresis of the labeled acetylcholine receptor showed that only the 40 000-dalton subunit contained the label. From these results it is concluded that the 40 000-dalton subunit represents a major component of the agonist binding site of the receptor.

Binding of n-alkyl sulphates to lysozyme in aqueous solution

Abstract: The binding of a homologous series of n-alkyl sulphates to lysozyme has been measured by equilibrium dialysis at 25°C. The binding isotherms show a concentration dependence attributable to aggregation of the protein–surfactant complexes. Both sodium n-dodecyl and n-decyl sulphates give binding isotherms characteristic of specific high energy interactions at low free surfactant concentrations. These are followed by non-specific cooperative binding. Sodium n-octyl sulphate interacts only cooperatively with lysozyme. The binding isotherms are discussed in terms of the binding potential concept of Wyman and are used to calculate an apparent Gibbs energy of binding per surfactant anion as a function of the number of surfactant anions bound.

The Receptors : a comprehensive treatise

Abstract: 1 Reconstitution of Membrane Transport Functions.- 1. Introduction.- 2. Reconstitution of Active and Passive Transport Systems.- 3. General Techniques of Reconstitution.- 3.1. Liposomes: Test Tubes with a Difference.- 3.1.1. Multilamellar Liposomes.- 3.1.2. Unilamellar Liposomes.- 3.2. Methods for Inserting Proteins into Liposomes.- 3.2.1. Cholate Dialysis.- 3.2.2. Sonication.- 3.2.3. Incorporation.- 3.2.4. The Use of Superstable Membrane Proteins.- 4. What We Can Learn from Reconstitution.- 4.1. Oxidative Phosphorylation.- 4.2. Ca2+-ATPase.- 4.3. (Na+ + K+)-ATPase.- 4.4. Acetylcholine Receptor.- 4.5. The Problem of Orientation.- 5. Reconstitution in Planar Bilayer Membranes.- 5.1. Sucrase-Isomaltase Complex.- 5.2. Acetylcholine Receptor in Planar Bilayers.- 5.3. Insertion of Whole Membrane Vesicles.- 5.4. Proton Pumps.- References.- 2 The Pharmacon-Receptor-Effector Concept: A Basis for Understanding the Transmission of Information in Biological Systems.- 1. Introduction.- 2. Biological Action.- 3. Receptors and Receptor Sites.- 4. Pharmacon-Receptor Interaction.- 5. Spare Receptors.- 6. Structure and Action.- 7. Accessory Receptor Sites.- 8. Steric Structure and Action.- 9. Selectivity in Action.- 10. Differentiation in Closely Related Receptor Types.- 11. Receptor Binding and Receptor Isolation.- 12. Dualism in Receptors for Agonists and Their Competitive Antagonists.- 13. The Aggregation-Segregation Concept.- 14. Dual Receptor Model.- 15. Combination of Pharmaca.- 16. The Slope of the Concentration-Effect Curves.- 17. The Allosteric Receptor Model.- 18. Binding and Displacement on Two or More Independent Classes of Receptor Sites.- 19. Two-Site Model.- 20. Reflection.- References.- 3 The Link between Drug Binding and Response: Theories and Observations.- 1. The Response to Acetylcholine-Like Drugs.- 1.1. Methods of Investigation of the Response.- 1.2. The Nature of the Response to Acetylcholine.- 1.3. The Response-Concentration Curve at Equilibrium.- 1.4. The Kinetics of the Response.- 1.4.1. Relationship between Methods of Studying Kinetics.- 1.4.2. Concentration-Jump Studies.- 1.4.3. Fluctuation Analysis.- 1.4.4. Voltage-Jump Relaxation Studies.- 1.5. Anesthetics, Local Anesthetics, and Channel Blocking.- 2. The Binding of Drugs to Acetylcholine Receptors.- 2.1. Methods for Investigation of Binding.- 2.2. Binding at Equilibrium.- 2.2.1. Cooperativity in Binding.- 2.2.2. Is There a Single Sort of Binding Site?.- 2.2.3. Binding to Junctional and Extrajunctional Receptors in Muscle.- 2.3. The Kinetics of Acetylcholine Binding.- 3. The Link between Drug Binding and Response.- 3.1. What Should a Mechanism Explain?.- 3.2. Some Mechanisms.- 3.3. The Concentration Dependence of Binding and Response at Equilibrium.- 3.4. The Nature of Efficacy, Partial Agonists, and Desensitization...- 3.5. Kinetics and Mechanism.- 3.5.1. What Does the Observation of a Single Time Constant Imply?.- 3.5.2. What Is the Rate-Limiting Step?.- 3.5.3. Concentration Dependence of Time Constants from Kinetic Studies.- 3.6. What Is the Origin of Voltage Dependence?.- 3.7. High Affinity Versus High Speed.- References.- 4 Kinetics of Cooperative Binding.- 1. Overview.- 2. General Introduction.- 3. Model I: koff as a Linear Function of Occupancy.- 3.1. Assumptions.- 3.2. Properties of the Model.- 3.2.1. Equilibrium.- 3.2.2. Association Curves.- 3.2.3. Dissociation Curves.- 3.3. Discussion.- 4. Application to the Insulin-Receptor System.- 4.1. The Controversy.- 4.2. Experimental Design.- 4.3. Simulation Results.- 4.4. Discussion.- 5. Model II: kon as a Linear Function of Occupancy.- 5.1. Introduction.- 5.2. The Model.- 5.3. Properties of the Model.- 5.3.1. Equilibrium.- 5.3.2. Association Curves.- 5.3.3. Dissociation Curves.- 5.4. Testing for Positive Cooperativity.- 6. General Discussion.- 7. A Guide to the Experimentalist.- Appendix A: Model I: Differential Equations and Solutions.- Appendix B: Addition of Fresh (Empty) Receptors.- Appendix C: Model II: Labeled Ligand Only.- Appendix D: Model II: Labeled and Unlabeled Ligands.- Appendix E: Optimization of Testing for Model II with ? > 0.- References.- 5 Distinction of Receptor from Nonreceptor Interactions in Binding Studies.- 1. Defining a Pharmacologic Receptor.- 2. Criteria for Receptor Interactions.- 3. The Problem of Relating Binding to Biological Responsiveness.- 4. Nonspecific Binding: Definition and Examples of Complications of Binding Data Analysis.- 5. Estimating the Affinity of the Unlabeled Ligand.- 6. Examples of Receptor-Like Nonreceptor Interactions.- 7. Conclusion.- References.- 6 Incorporation of Transport Molecules into Black Lipid Membranes.- 1. Introduction.- 2. Methodology.- 2.1. Formation and Composition of BLMs.- 2.2. Electrical Properties of BLMs.- 3. Mechanisms of Ion Permeability.- 3.1. Carriers.- 3.2. Channel Formers.- 4. Models of Interactions of Proteins with BLMs.- 5. Ionophorous Properties in BLMs of Functional Transport Molecules.- 5.1. Ca2+-ATPase: Dissection of a Transport System.- 5.2. (Na+ + K+)-ATPase.- 5.3. The Acetylcholine Receptor.- 6. The BLM as a Test System for Ionophorous Function of Isolated Membrane Proteins.- 6.1. Mitochondrial Membrane Proteins.- 6.2. Red Blood Cell Membrane Proteins.- 6.3. Gastric Mucosal Membrane Proteins.- 6.4. Dopamine-?-Hydroxylase.- 6.5. Rhodopsin.- 6.6. Immune Cytotoxic Factors.- 7. Coda.- References.- 7 Visualization and Counting of Receptors at the Light and Electron Microscope Levels.- 1. Receptors at the Cell Membrane.- 1.1. Introduction.- 1.2. Information Required on the Distribution of Receptors.- 2. The Labeling of Receptors for Localization.- 2.1. Approaches.- 2.2. Methods of Labeling and Visualizing Receptors.- 2.2.1. Autoradiography.- 2.2.2. Electron-Dense Label Attachment.- 2.2.3. Enzymatic Reaction Product Markers.- 2.2.4. Fluorescent Markers.- 2.2.5. X-Ray Microanalysis.- 2.3. Ligands for Receptor Labeling.- 2.3.1. Selection of Primary Ligands.- 2.3.2. Ligands Available for Receptor Tracing.- 3. Cell and Tissue Autoradiography.- 3.1. Problems of Application of a Labeled Ligand.- 3.1.1. Mode of Application.- 3.1.2. Nonspecific Labeling.- 3.1.3. Tissue Processing for Autoradiography of Receptors.- 3.1.4. Application to a Pseudo-Irreversible Reaction at a Receptor.- 3.2. Autoradiographic Methods.- 3.2.1. Treatments in Aqueous Media.- 3.2.2. Dry-Mount Methods.- 3.3. Interpretation of EM Autoradiographic Data on Receptors.- 3.3.1. Assignment of Silver Grains in Autoradiographs to Most Probable Locations of the Labeled Receptors.- 3.3.2. Calculation of Receptor Density.- 3.3.3. Isotopes and Resolution.- 3.4. Applications to Synaptic Receptors.- 4. Counting Receptors per Cell or per Synapse.- 4.1. Light Microscope Autoradiography of Receptors.- 4.2. Absolute Enumeration of Total Receptors.- 4.3. Direct Determination of Receptor Occupancy Relations.- 5. Electron Microscope Methods for Visualization of Receptors.- 5.1. Peroxidase Cytochemistry of Receptors.- 5.1.1. Peroxidase Methods.- 5.1.2. Applications of Peroxidase Cytochemistry to Receptors.- 5.2. Tissue Preservation for Immunocytochemistry of Receptors.- 5.3. Ferritin Labeling.- 5.4. Other Labels Applicable for Transmission and Scanning EM Studies of Receptors.- 6. Fluorescence Marker Methods.- 6.1. Fluorescence Labeling.- 6.2. Application of Fluorescent Labeling to Receptors.- 7. Possibilities of Quantitation of Receptors in Immunocytochemical and Other Nonradioisotopic Techniques.- 7.1. Quantitation in Electron-Dense Marker Techniques.- 8. Conclusions.- References.- 8 Problems and Approaches in Noncatalytic Biochemistry.- 1. Introduction.- 2. Measurement.- 2.1. Histological Techniques.- 2.2. Physical Separation: General Considerations.- 2.3. Equilibrium Dialysis.- 2.4. Other Physical Separations.- 2.5. Negative Binding.- 2.6. The Magnitude of the Off-Time.- 3. Relation of in Vivo to in Vitro Properties.- 3.1. Reversibility.- 3.2. Location.- 3.3. Specificity.- 3.4. Dissociation Constants.- 3.5. Detergents.- References.

The Relipidation of Delipidated Na,K‐ATPase

Abstract: Enzymatically inactive, delipidated Na,K-ATPase from dogfish rectal glands was titrated with dioleoylphosphatidylcholine and with dioleoylphosphatidylethanolamine The process of relipidation has the following characteristic properties Enzymatic activities reappear independently of each other: first the phosphatase, then the ATPase The properties of the phosphatase regenerated depend on the ratio of lipid/protein used; the ATPase seems to be independent of this ratio The simplest model that is consistent with the above results and with the shapes of the titration curves, has the following requirements Firstly, the enzyme is composed of two subunits that, as far as lipid binding is concerned, are identical and independent of each other Secondly, lipid adds onto the enzyme as preformed clumps of 25 molecules of phosphatidylcholine or 18 molecules of phosphatidylethanolamine Thirdly, each subunit binds two clumps of lipid, and binding shows positive cooperativity Fourthly, when either subunit becomes saturated with lipid, the enzyme exhibits one form of phosphatase Fifthly, when both subunits are saturated with lipid, the enzyme exhibits a second form of phosphatase and ATPase The data and their analysis according to this model lead to the suggestion that Na,K-ATPase is a functional dimer, the interaction between subunits being influenced by the Na+ and K+ concentrations in the medium: K+ favouring the functional independence of the subunits and Na+favouring their functional interaction

Multi-subsite receptors for multivalent ligands. Application to drugs, hormones, and neurotransmitters.

Abstract: Detailed structure-activity relationships of many drugs and hormones indicate that ligand-receptor binding involves the interaction of several functional regions on the ligand with complementary receptor "subsites." However most hormone receptor binding models have been based on a simple bimolecular reaction obeying the mass action law. We present a quantitative reinterpretation in pharmacological terms of the theory of flexible polyvalent ligand binding. The model explains: 1) the occurrence of complex binding isotherms showing both apparent heterogeneous and cooperative binding sites; 2) bell-shaped dose response curves; 3) the properties of full and partial agonists; 4) how a given antagonist can be either "competitive" or "noncompetitive" depending on concentration used. The classical simple bimolecular interaction between drug and receptor is a limiting case of the model, when steric hindrance completely prevents multiple receptor occupancy, or when the ligand and the receptor interact in an all-or-none mode.

The heterogeneity of nerve growth factor receptors.

Abstract: Chick embryonic sensory ganglia cells have two specific nerve growth factor receptors, site I and site II receptors, whose binding affinities differ by two orders of magnitude. As judged by both steady state binding and kinetic data, the two receptors behave independently. The rate of dissociation of the labeled nerve growth factor from site I receptors is increased in the presence of unlabeled nerve growth factor even when its concentration is below that of the labeled growth factor used to equilibrate the cells, a phenomenon which cannot be explained by negative cooperativity. Site I receptors are present only on neurons while site II receptors are present on both neurons and nonneuronal cells. At the concentration of nerve growth factor which produces half maximal stimulation of neurite outgrowth 8% of site I and 0.1% of site II receptors are occupied. This occupancy of site II receptors falls to about 0.01% with bisdesarginine beta nerve growth factor, a derivative which is as biologically active as the unmodified factor but which binds with lower affinity to site II receptors. These data support the idea that interaction of nerve growth factor with site I receptors is responsible for the initiation of neurite outgrowth.

Allosteric cooperative interactions among redox sites of Pseudomonas cytochrome oxidase

Abstract: Anaerobic reductive spectrophotometric titrations of Pseudomonas aeruginosa cytochrome oxidase were performed. Both types of hemes (C and D) of the dimeric enzyme were monitored. The reduction process was found to involve cooperative allosteric and spectroscopic interactions between the two subunits. The model fitting the data best involves the following features. (1) The redox potential of heme C is about 60 mV higher than that of heme D. (2) In the electron uptake, a positive cooperativity of about 30 mV exists between the two D-type hemes residing in the two subunits. (3) A negative cooperativity of the same magnitude (30 mV) is found between the two C-type hemes bound to two subunits. (4) No interaction was found between heme C and D in the same subunit or in the different subunits. (5) It is suggested that the reduction of the heme, of each kind, has about twice the spectral change compared to that observed upon reduction of the second one. The possible significance of this model for the mechanism of action of the enzyme is discussed

Contribution of negative cooperativity to the thyrotropin-receptor interaction in normal human thyroid: kinetic evaluation.

Abstract: The kinetics of 125I-labeled thyrotropin (125I-TSH) binding to human thyroid receptors are presented. At pH 6.0, binding was maximal (30--35%) and there was one class of binding sites [Kd = 6.8 X 10(-9) M; binding capacity (Ro) = 57 pmol/mg of protein]. At pH 7.4, Scatchard plots of binding were nonlinear, indicating either a single class of negatively cooperative sites (Kd = 3.7 X 10(-9) M; Ro = 26 pmol/mg of protein) or, alternatively, independent high- (Kd = 5.0 X 10(-10) M; Ro = 3 pmol/mg of protein) and low-affinity (Kd = 1.7 X 10(-8) M; Ro = 26 pmol/mg of protein) binding sites. The role of negative cooperativity was evaluated from the rates of association and dissociation at pH 7.4. The kinetically determined binding constants (Kd = 1.7 X 10(-11) M; Ro = 2 pmol/mg of protein) were more similar to those determined for the high-affinity component than to those predicted from the negative cooperativity model. Dissociation of bound TSH was independent of initial site occupancy over a 40-fold range, corresponding to a 100-fold range of free TSH concentration. The dissociation rate of 125I-TSH was enhanced by unlabeled TSH to a similar degree, irrespective of initial binding site occupancy. Because the negative cooperativity model does not accommodate these data, it is concluded that TSH receptors in human thyroid behave kinetically and at equilibrium as a single class of high-affinity sites up to TSH concentrations well above the physiological range.

Conformational Transition of Poly(L-glutamic acid) in Aqueous Decylammonium Chloride Solution

Abstract: The cooperative binding of decylammonium chloride to poly(L-glutamic acid) (PLG) was studied by the potentiometric measurement of the binding isotherm at pH 7.9. Circular dichroism spectra were also measured as a function of the degree of binding of surfactant ion (x). It was shown that the coil to α-helix transition of PLG-surfactant complex takes place with a change in x. PLG did not undergo an appreciable conformational change in the range of x below 0.55. But the helical content increased suddenly with further increase in x. An abrupt increase in the helical content of PLG-surfactant complex can be well interpreted in terms of the hydrophobic interaction among bound surfactant ions. On the basis of the theoretical analysis of the cooperative binding isotherm, it was concluded that the formation of a micelle-like cluster consisting of at least eight surfactant ions is required for the stabilization of a surfactant induced helical structure.

Numerical analysis of binding studies: A direct procedure avoiding the pitfalls of a scatchard analysis of equilibrium data for unknown binding models

Abstract: It is shown on theoretical grounds that the straightforward analysis of binding data according to Scatchard may lead to erroneous results, especially when more complicated binding schemes are involved. We have demonstrated this point by presenting Scatchard plots with slight variation of experimental parameters. These inherent difficulties of Scatchard analyses can be avoided by applying a direct procedure. We have developed a program, which compares the measured quantity and the theoretical value directly and which considers the following binding models: 1. (i) independent equivalent binding of n ligands; 2. (ii) independent unequivalent binding of 2 ligands; 3. (iii) positive or negative cooperative binding of 2 ligands. Other binding schemes can easily be implemented. We have used this procedure for the evaluation of equilibrium data on the complex formation of tRNA-Tyr and tyroyl tRNA synthetase from E. coli in terms of different binding models.

Ligand Competition Curves as a Diagnostic Tool for Delineating the Nature of Site-Site Interactions: Theory

Abstract: A few molecular models have been developed in recent years to explain the mechanism of cooperative ligand binding. The concerted model of Monod, Wyman and Changeux and the sequential model of Koshland, Nemethy and Filmer were formulated to account for positively cooperative binding. The pre-existent asymmetry model and the sequential model can account for negatively cooperative ligand binding. In most cases, however, it is virtually impossible to deduce the molecular mechanism of ligand binding solely from the shape of the binding isotherm. In the present study we suggest a new strategy for delineating the molecular mechanism responsible for cooperative ligand binding from binding isotherms. In this approach one examines the effect of one ligand on the cooperativity observed in the binding of another ligand, where the two ligands compete for the same set of binding sites. It is demonstrated that the cooperativity of ligand binding can be modulated when a competitive ligdnd is present in the protein-ligand binding mixture. A general mathematical formulation of this modulation is presented in thermodynamic terms, using model- independent parameters. The relation between the Hill coefficient at 50% ligand saturation with respect to ligand X in the absence, h(x), and in the presence of a competing ligand Z, h(x,z), is expressed in terms of the thermodynamic parameters characterizing the binding of the two ligands. Then the relationship between h(x) and h(x,z), in terms of the molecular parameters of the different allosteric models, is explored. This analysis reveals that the different allosteric models predict different relationships between h(x,z) and h(x). These differences are especially focused when Z binds non-cooperatively. Thus, it becomes possible, on the basis of ligand binding experiments alone, to decide which of the allosteric models best fits a set of experimental data.

AMP Deaminase from Baker′s Yeast--Kinetic and Molecular Properties

Abstract: The kinetic and molecular properties of AMP deaminase [AMP aminohydrolase, EC 3.5.4.6] purified from baker's yeast (saccharomyces cerevisiae) were investigated. The enzyme was activated by ATP and dATP, but inhibited by Pi and GTP in an allosteric manner. Alkali metal ions and alkaline earth metal ions activated the enzyme to various extent. Kinetic negative cooperativity was observed in the binding of nucleoside triphosphates. Kinetic analysis showed that the number of interaction sites for AMP (substrate) and Pi (inhibitor) is two each per enzyme molecule. The molecular weight of the native enzyme was estimated to be 360,000 by sedimentation equilibrium studies. On polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate, the enzyme gave a single polypeptide band with a molecular weight of 83,000, suggesting that the native enzyme has a tetrameric structure. Baker's yeast AMP deaminase was concluded to consist of two "promoter" units which each consist of two polypeptide chains with identical molecular weight.

Complex formation of acridine orange with single-stranded polyriboadenylic acid and 5'-AMP: cooperative binding and intercalation between bases.

Abstract: The binding of acridine orange to single-stranded polyribonucleic acid at low polymer to dye ratios exhibits cooperative behavior of the kind observed with other simple polyanions. It is thus attributed to electrostatic interaction between polymer and stacked dye molecules. At higher polymer to dye ratios, however, distinct deviations from the predictions of the basic theory occur. These are interpreted by additional non-cooperative binding of acridine orange to the bases of the polymer subunits owing to dye-base stacking. This effect is studied also with 5′-AMP monomers where it likewise leads to complex formation. Both systems are investigated experimentally by means of the changes produced in the dye spectrum. Based on quantitative analyses the equilibrium constants of both systems are evaluated and discussed. They indicate a sandwich-type of intercalation of dye between two bases of the single-stranded polymer.

Role of metal cofactors in enzyme regulation. Differences in the regulatory properties of the Escherichia coli nicotinamide adenine dinucleotide phosphate specific malic enzyme, depending on whether magnesium ion or manganese ion serves as divalent cation.

Abstract: A number of differences in the kinetic and physical properties of the Escherichia coli nicotinamide adenine dinucleotide phosphate (NADP+) dependent malic enzyme have been found, depending upon whether Mg2+ or Mn2+ served to fulfill the divalent cation requirement. The velocity-NADP+ and velocity-cation saturation curves exhibit a simple hyperbolic response in the presence of either metal cofactor, but the affinity for NADP+ (and malate) as well as the Vmax is increased in the presence of Mn2+. The high affinity of the enzyme for Mn2+ coupled with the increased affinity for substrates indicates that Mn2+ is the preferred cofactor in vitro. With either Mg2+ or Mn2+ as cation, the velocity-malate saturation curves in the absence of effectors are complex at pH 7.45, indicating varying combinations of apparent positive and negative cooperative behavior. Greater initial positive cooperative behavior between malate binding sites is observed with Mg2+ as cation. The enzyme appears to be equally sensitive to inhibition by the allosteric inhibitors reduced nicotinamide adenine dinucleotide (NADH) and oxaloacetic acid (OAA) in the presence of either cation, but the interaction between malate binding sites, in the presence of effectors, varies significantly with the choice of metal cofactor. The inhibitor NADH increases the interaction between malate binding sites in the presence of Mn2+ but has little effect on subunit interaction in the presence of Mg2+. The inhibitor OAA increases the interaction between malate binding sites in the presence of both cations, with increased positive cooperativity observed with Mn2+ but increased negative cooperativity with Mg2+. The kinetic data can be explained by a model involving sequential ligand-induced conformational changes of the enzyme, resulting in a mixture of apparent positive and negative cooperative behavior. Alternative explanations involving different classes of noninteracting binding sites or different enzyme forms are also considered. The metal cofactors, Mg2+ and Mn2+, appear to stabilize two distinct conformational states of the enzyme which differ in response to varying substrate and effector concentrations. Altered conformational states of the enzyme in the presence of the two cations are further substantiated by proteolytic digestion studies with the homogeneous enzyme. The results are strikingly similar to previous results reported on the nicotinamide adenine dinucleotide (NAD+) dependent malic enzyme and the NAD+-dependent isocitrate dehydrogenase, supporting the suggestion that metal cofactors function as regulatory entities.

Effect of interchain disulfide bond on hapten binding properties of light chain dimer of protein 315

Abstract: The hapten binding characteristics of the covalent light chain dimer, derived from the murine IgA secreted by plasmacytoma MOPC-315, to two nitroaromatic compounds, epsilon-N-(2,4-dinitrophenyl)-L-lysine and 4-(alpha-N-alanine)-m-nitrobenz-2-oxa-1,3-diazole, were investigated by differential spectroscopic titrations The binding curves for both haptens were found to display sigmoidity similar to that reported earlier for the reduced and alkylated dimer held together by noncovalent bonds only However, the presence of the interchain disulfide bond in the covalent dimer was found to cause marked changes in its binding properties The data, like those obtained for the noncovalent dimer, fit the allosteric model of Monod, Wyman, and Changeux in which binding of the first hapten to the dimer causes a conversion of both sites of the protein molecule from a lower to a higher affinity conformation However, the binding parameters show that both the affinity and the positive cooperativity in the interaction between haptens and the covalent dimer are significantly enhanced The differences in the parameters of the binding and of the allosteric transition caused by the presence of the interchain disulfide bond demonstrate the existence of longitudinal interactions in immunoglobulin derivatives These properties of the light chain dimer make it a potential model for the receptors present on thymus-derived lymphocytes