Showing papers on "Cooperative binding published in 1972"

Ethidium Bromide as a Cooperative Effector of a DNA Structure

Abstract: A salt-induced cooperative conformational transition of a synthetic DNA, poly(dG-dC), is reversed by addition of ethidium bromide. Binding of the dye at high salt concentrations is highly cooperative. Circular dichroism spectra of the complex and the kinetic data support a model for this cooperative binding that is formally equivalent to the “allosteric” one proposed for oligomeric proteins by Monod et al. Thus, double-helical DNA of at least one defined sequence can undergo a cooperative conformational change in solution, with simple salts and drug molecules as antagonistic effectors. Such transitions may be involved in regulatory phenomena operating directly at the level of nucleic acid structure.

Molecular analysis of the membrane attack mechanism of complement

Abstract: The molecular arrangement of the membrane attack mechanism of complement was explored. The molar ratios of the components within the C5-9 assembly on the target cell surface were determined using human complement proteins in highly purified and radiolabeled form. With the aid of monospecific complement antisera it was possible to probe the spatial relationships between the components of the assembly. C5 and C6, in the presence of C7, were bound to EAC1-3 in equimolar quantities irrespective of the amounts and the relative proportions of C5, C6, and C7 offered. The amount of C8 bound to EAC1-7 increased with input and at saturation of all C8 binding sites the molar ratio of bound C8/bound C5 approached 1.0. Uptake of C9 by EAC1-8 increased with input and at saturation of all C9 binding sites the molar ratio of bound C9/bound C8 became 6.0. However, calculations suggest that the binding of three C9 molecules to one C8 molecule is sufficient to achieve a full hemolytic effect. Evidence was obtained indicating that binding and hemolytic function of C9 depends upon cooperative interaction of multiple C9 molecules. Binding of C8 to EAC1-7 and the generation of hemolytic C8 sites were inhibited by antibody to either C5, C6, or C7. Uptake of C9 by EAC1-8 and the generation of hemolytic C9 sites were strongly inhibited by anti-C8 and to a lesser degree by anti-C5. Binding of C9 (but not hemolysis) was also reduced by antibody to C6 or C7. The data are consistent with the concept that the fully assembled membrane attack mechanism of complement consists of a decamolecular complex: a trimolecular arrangement composed of C5, C6, and C7 forms the binding site for one C8 molecule which in turn furnishes binding sites for six C9 molecules, saturation of three sites apparently being sufficient for expression of full cytolytic activity of the complex. This work made it possible to design a simple molecular model.

Receptors on immunocompetent cells : iv. direct measurement of avidity of cell receptors and cooperative binding of multivalent ligands

Abstract: The interaction of antigen with specific, cell-associated receptors was measured in thermodynamic terms. The binding of (125)I-labeled 2,4-dinitrophenyl guinea pig albumin (DNP(16)GPA-(125)I) to lymphocytes from guinea pigs immunized to DNP(16)GPA is a temperature-dependent, reversible process. Measurement of association and dissociation rates of antigen-receptor complexes permits calculation of antigen-cell binding constants. These may also be calculated by equilibrium-binding techniques. Although differences in the constants calculated in these two ways exist, a clear increase in avidity of cell receptor for antigen occurs in the course of the immune response. This change in receptor avidity provides evidence that the time-dependent change in affinity of serum antibody (maturation) indeed has a cellular basis. The magnitude of the equilibrium constant is, in part, due to binding of more than one DNP group per molecule of antigen. Thus, multivalent ligands bind more effectively to cell receptors than univalent or paucivalent ligands when measured by the number of antigen molecules bound, the dissociation rate of antigen-receptor complexes, and in the relative capacity to inhibit a standard multivalent ligand (DNP(16)GPA-(125)I) from binding.

The binding of oxidized and reduced nicotinamide adenine-dinucleotide to yeast glyceraldehyde-3-phosphate dehydrogenase.

Abstract: The binding of NADH to yeast glyceraldehyde-3-phosphate dehydrogenase has been compared with that of NAD+ under otherwise identical conditions (i.e., pH 8.5 and 20 °C). The binding of NAD+ is weakly sigmoidal. Kinetic data obtained with chemical relaxation and rapid-mixing techniques are consistent with the concerted mechanism for the cooperative binding of NAD+. In particular, the apo-enzyme consists of a mixture of two states (90% with low affinity and 10% with high affinity for NAD+) at equilibrium with each other. In contrast to NAD+, the binding of NADH is hyperbolic. Only one relevant relaxation process was observed in the 0.1 msec range. Since the reciprocal relaxation time increases with increasing concentration of NADH, the reaction can be identified with an elementary binding reaction. NADH and NAD+ compete for the same binding site. Rapid displacement of NADH by NAD+ has been used as a diagnostic tool for the ability of NADH to shift the allosteric preequilibrium. This technique and the rapid titration of sulfhydryl groups with 5,5′-dithio-bis-(2-nitrobenzoic acid) establish that NADH binds with equal affinity to both allosteric states of yeast glyceraldehyde-3-phosphate dehydrogenase. This finding accounts for the relatively simple relaxation spectrum of NADH binding. These and other data from the literature can be rationalized on the basis of separate subsites for the adenine and nicotinamide moieties of NAD+, the accessibilities of which differ in the two allosteric states. The possibility of a regulation of the fraction of enzymically active yeast glyceraldehyde-3-phosphate dehydrogenase by the NADH/NAD+ ratio is discussed.

Observation of Cooperative Ionizations in Hemoglobin

Abstract: 19F-Nuclear magnetic resonance studies of specifically fluorinated hemoglobin derivatives have been used to determine the apparent pKa of the histidine β146 imidazole in deoxyhemoglobin. The titration of this residue was found to be abnormally sharp, particularly in the presence of diphosphoglyceric acid. The explanation advanced for this unusual titration curve may have implications for the mechanism of cooperative ligand binding. The possible role of such ionizations is discussed in light of some chemical evidence that the cooperative binding process is governed to a greater extent by internal nonpolar forces than by electrostatic interactions of exposed groups.

Subunit interactions in allosteric control

Abstract: Publisher Summary This chapter discusses about subunit interactions in allosteric control. Cooperative effects were originally discovered in hemoglobin and have been shown to be a characteristic of most regulatory proteins. To understand the ligand-induced changes of a protein molecule it is necessary to develop diagnostic methods. It is apparent that a quantitative evaluation of the intrinsic constants of ligand binding can lead to a diagnosis between models and therefore can provide insight into the strength of the individual molecular parameters. Ligand-induced conformational changes are required to explain negative cooperativity apparent in many proteins. They are required to explain the physical changes indicated in a positively cooperative system like hemoglobin. It appears that many other processes—the peeling of a repressor from DNA, the change in a transport protein on a membrane, the reaction of a nerve receptor—are similar in nature and involve induced conformational changes.