Showing papers on "Cooperativity published in 1973"

Alteration of the Relative Stability of dA·dT and dG·dC Base Pairs in DNA

Abstract: Several small alkylammonium ions can eliminate, or even reverse, the usual dependence of the DNA transition temperature on base composition. For example, in 3 M tetramethylammonium chloride, or 2.4 M tetraethylammonium chloride, DNAs of different base compositions all melt at a common temperature, and with a greatly decreased breadth of transition reflecting only the sequence-independent components of melting cooperativity. At still higher concentrations of such additives, dG·dC-rich DNAs melt at lower temperatures than dA·dT-rich molecules. Circular dichroism spectra show that these additives alter the structure of the DNA double helix very little at room temperature. This differential (base-specific) effect on helix stability is investigated with several small additives related to the tetraalkylammonium ions. Additives larger than tetraethylammonium ion have little differential effect on helix stability. Preferential binding of ions to dA·dT base pairs, requiring fit into a “groove” of DNA, is consistent with these data and with equilibrium binding studies. These differential effects can be distinguished from general destabilizing effects, which are independent of specific features of macromolecular conformation or chemistry. Possible experimental uses of this ability to alter the base-composition-dependent components of the stability of the DNA helix are discussed, as well as the insight this phenomenon provides into the molecular basis for the differential stability of dA·dT and dG·dC base pairs.

The Cooperativity of γ-Aminobutyric Acid Action on the Membrane of Locust Muscle Fibers

Abstract: Dose-response curves of the action of γ-aminobutyric acid to increase the conductance of postsynaptic membrane were measured in fibers of the flexor tibialis muscle of the metathoracic leg of Locusta migratoria . The sigmoid shape of the curves is interpreted in terms of the cooperative binding of more than 1 γ-aminobutyric acid molecule to the receptor. The "function of state," which describes the particular case in which receptor activation is induced only after the binding of more than 1 ligand molecule, is compared with some other formulations of cooperative binding, and is shown to give a better fit to the experimental data. This function uniquely predicts that the limiting Hill slope at small concentrations equals n , the number of molecules required to activate a receptor. In the present experiments n appears to be 3.

Apparent cooperativity in radioimmunoassay of human chorionic gonadotropin.

Abstract: Eight of 11 guinea pig antisera to human chorionic gonadotropin (hCG) examined in radioimmunoassay showed enhanced binding of labeled hCG after addition of small increments of unlabeled hCG. Gel chromatography of one antiserum revealed that this effect was attributable to antibodies on the 7S class. Mild papain digestion of the anti—hCG produced a modified antibody, possibly divalent F(ab)2, which showed enhanced binding and univalent Fab, which did not. These data indicated that the effect was related to the multivalence of antibodies, and tended to exclude a variety of other possible explanations. Various binding analyses suggested that the data were consistent with positive cooperativity between the two combining sites of a small population of antibodies. However, it is not clear whether the apparent cooperativity of the multivalent antibodies results from allosterism, or is related to another mechanism of enhanced binding. (Endocrinology 92: 1250, 1973)

Tryptophanyl-Transfer Ribonucleic-Acid Synthetase from Beef Pancreas

Abstract: Binding of tryptophan, tryptamine and ATP to tryptophanyl-tRNA synthetase was studied by equilibrium dialysis experiments. There are two binding sites per mole of enzyme both for tryptophan and for tryptamine. Ks for tryptophan is 0.95 μM and for tryptamine is 1.8 μM. In the case of ATP no binding could be measured over the range of concentrations examined. The Scatchard plots of tryptophan and tryptamine binding do not shown any cooperativity between the subunits. Dissociation of the dimeric enzyme was studied at very low protein concentration. Upon dilution of the enzyme both the [32P]PPi-ATP isotope exchange activity and the tRNA charging activity are lost simultaneously. Kinetic evidence demonstrates that the inactivation is primarily due to the dissociation of the active dimer into inactive monomers. The dissociation is strongly promoted by alkaline pH and is inhibited by the presence of tryptophan or tryptophanyladenylate but not by that of tRNA. The dissociation constant of the dimer-monomer equilibrium at pH 8.5 is 15 nM at 25 °C. The dissociation form is more susceptible to denaturation than the dimeric species. The results reported in the paper suggest that even if there are not binding interactions between the subunits, the active conformation of the enzyme depends on their associated state.

Theory of Radioimmunoassays and Hormone-Receptor Interactions:

Abstract: Since antibody and some hormone receptor molecules are known to possess two binding sites, it is possible that the intrinsic affinity constant for the “second site”, after one site is filled, is greater than the intrinsic affinity constant for the first site; this would result in “cooperative” or apparent “allosteric” effects. The mathematical theory of “cooperative” binding systems, both at equilibrium and in the transient state, has been applied to radioimmunoassay systems. The effects of affinity constants, antibody concentration, and “tracer” concentration, have been examined in several coordinate systems. Further, the kinetics of these reaction systems have been simulated. This theory should be useful in design and analysis of experiments to test for the presence of “cooperativity” effects.

The conformational stability of ribosomal proteins L7 and L12 from E. coli. I. Circular dichroism study of the changes induced by ionic strength and solvents.

Abstract: Ribosomal proteins L7 and L12 are the only acidic proteins found on the 50S ribosomal subunit of Escherichia coli. The effect of ionic strength, helix-promoting solvents and denaturating agents on the conformation of these proteins has been studied. It has been established that the helicity of L7 and L12 proteins (approx. 45–50% α helix) can be increased to 60–70% when they are exposed to helix-promoting solvents such as methanol or ethanol in the presence of 0.1M salt. High ionic strength by itself was without any effect on the conformation of the proteins. However, the solvent, 2,2,2-trifluoroethanol increased the content of α helices up to 80% even in the absence of salt. Denaturating agents like urea (6M) or guanidine HCl (6M), decreased the content of the ordered structure below 20%. All conformational changes induced by salt or solvents were completely reversible and characterized by a broad transition showing a low degree of cooperativity. This might indicate the presence of discrete segments with variations in amino acid sequences and ordered structures with different stabilities.

The cooperative interactions of complementary synthetic macromolecules in solutions

Abstract: Interactions between linear synthetic macromolecules containing complementary functional groups (for example, basic and acidic) have a well-expressed cooperative character and result in polymer-polymer complexes being formed. The cooperativity shows itself in a strong shift of the equilibria towards complex formation compared to the low molecular analogues, in the critical character of the dependence of the stability of the polycomplexes upon the chain lengths of the components, and also in the peculiarities of the distribution of oligomers along macromolecular templates. Complex formation between macromolecules is a structure-sensitive reaction. Sometimes it is accompanied by conformational transitions of polymer reagents. For example, one of the macromolecules may be ‘forced’ to acquire a conformation which it would not assume in the given conditions in the absence of the polymer partner (this is exemplified by ionogenic polypeptides altering the secondary structure). In most cases complex formation results in compact tertiary structures which seem to play an important role in the stabilization of the complexes.

Cooperative Ligand Binding by Ferrihemoglobin

Abstract: Reactions of human ferrihemoglobin with azide and fluoride ions have been reinvestigated, particularly with a view to compare the effect of pH on the Hill coefficient and to study its dependence on observation wavelength. The results at pH 6 (with initial high-spin aquo form) show the absence of cooperativity for the fluoride reaction but the existence of significant co-operativity for the azide reaction, in agreement with the conclusions drawn by some previous authors. At pH 9, where the ferrihemoglobin is an equilibrium mixture of high and low-spin states, the same reactions give different Hill coefficients; cooperativity appears for fluoride binding and diminishes for the azide reaction. The possible significance of concomitant changes of iron spin state and the appearance of cooperativity in hemoglobin reactions is discussed.

Polyelectrolyte Behaviour of Phosvitin

Abstract: The results of spectroscopic and microcalorimetric measurements on dilute aqueous solutions of phosvitin are described Our infrared and circular dichroism spectra show that the phosphoprotein chains may assume either random-coiled or partially helical or β-structures depending upon the pH The transition to the low pH β-form occurs with a distinct cooperative character The strongly interdependent ionization of fixed charges densely spaced along phosvitin chains, typical of highly charged polyelectrolytes, is well illustrated by the calorimetric enthalpy of protonation data The interaction of phosvitin with acridine orange in dilute aqueous solution (pH ∼5) has been studied by means of spectral and equilibrium dialysis experiments For relatively low polymer to dye concentration ratios, bound acridine orange exhibits γ-type spectrum thus suggesting aggregation of dye molecules onto the protein Gradual decrease in the extent of bound acridine orange aggregation is produced by increasing the concentration of the protein; the monomerically bound form is hardly obtained, however, even at relatively high phosvitin concentrations Binding curves reveal a cooperativity character for the dye-protein interaction It is suggested that about 60% only of phosphomonoester side groups of phosvitin are available for acridine orange binding, these being characterized by a high “stacking” coefficient The results are tentatively discussed on the basis of present information on phosvitin chemical composition and conformation

The effect of bifunctional and monofunctional azo-dye reagents upon the oxygen-binding mechanism of human hemoglobin.

Abstract: Both p-bis-iodoacetamido-2,2′-dicarboxyazobenzene and 4-iodoacetamido-2,3′-dicarboxyazo-benzene were reacted with the accessible SH-groups of human hemoglobin tetramers. The mono-functional azo dye produced the expected, and uniformly modified hemoglobin, in which both cysteines-β93 were alkylated. The bifunctional dye gave rise mainly to a symmetrically modified hemoglobin, forming cross-links within the two β-chains between cysteine-93 and lysine-82. A small amount of a second, asymmetrical hemoglobin derivative could be isolated, carrying the same cross-link in one β-chain, while the second cross-link ran from one cysteine-β93 to the histidine in position 45 of the neighbouring α, chain. Homotropic cooperativity of oxygen binding was reduced in all these derivatives of hemoglobin. Both cross-linked hemoglobins possessed an oxygen-binding curve with a Hill coefficient of 1.0–1.2. For the hemoglobin reacted with mono-functional dye this coefficient varied between 1.65 and 1.72. Its Bohr effect amounted to 0.3, while the cross-linked hemoglobins showed no Bohr effect.

Cooperative binding of oxygen to hemoglobin: analysis of accurate equilibrium binding data.

Abstract: Accurate equilibrium binding data for the oxygenation of hemoglobin are used (a) to show that various models for cooperativity are inconsistent with the best available experimental data, (b) to determine the equilibrium constants for binding of 2,3-diphosphoglycerate to hemoglobin molecules in intermediate stages of oxygenation, and (c) to deduce a mechanism for allosteric effects in hemoglobin which is consistent with the best available experimental data. The total free energy of cooperativity is defined and discussed.

On the theory of ion transport across the nerve membrane, VII. Cooperativity between channels of a large square lattice.

Abstract: The exact kinetics of a 10 × 10 Ising system (periodic boundary conditions) with two-state channels arranged in a square lattice was studied by computer simulation. With all three values of the cooperativity parameter used, no induction in the K+-current curve was obtained. This confirms one of our previous conclusions concerning K+ channels in the squid axon membrane: models with interacting channels arranged in a twodimensional lattice (Adam's model) are apparently excluded. Other topics included: equilibrium properties; short-range pair correlation functions; phase transition.

Molecular conformation and cooperativity in hemoglobin

Abstract: One of the most intensively studied problems in biochemistry is the nature of the molecular processes which give rise to cooperative ligand binding in hemoglobin. The most precise ligand binding data available can be shown to be consistent with several mechanistic models which are based on entirely different (but kinetically indistinguishable) concepts of molecular Elucidation of the crystal structures5 of the fully liganded and unliganded forms was an important advance toward understanding of the molecular forces involved in stabilizing two forms of the protein, but since the intermediate stages of the ligation process are not accessible to crystallographic analysis, knowledge of the endpoints does not resolve a number of questions about the detailed mechanism of conversion. Spectroscopic studies in solution are beginning to yield some information on the intermediate stages of ligand binding. The nuclear magnetic resonance (nmr) studies of Shulman and coworkers on “artificial” intermediates and the work of Ogata and McConnell 6 on binding of allosteric effectors have demonstrated changes in one region of the molecule that are consistent with a single concerted transition between low and high affinity forms, but do not preclude the presence of forms of intermediate structure and ligation at some stages of the reaction path. All of the mechanistic models being discussed contain the basic premise that at least two structures of the protein exist which differ in ligand affinity. The differences between models arise over the number of discrete structures which exist, the role (if any) played by the ligand itself in the transition, and the relative energetic importance of various regions of the molecule to stability of various forms. The investigations described here are fluorine and phosphorus nmr studies of conformational changes in two regions of the hemoglobin molecule at various stages of fractional ligation. Nmr studies of a fluorinated label situated at cysteine 93 in the B chains yield information on tertiary structural changes in a crucial region of the a,& interface, while 3lP-nmr studies of the binding of diphosphoglyceric acid (DPG) permit detection of the quaternary structural changes influencing the DPG binding site. Both types of studies were conducted with concurrent measurements of optical absorbance at each ligand pressure,

A calorimetric study of a polymer–monomer complex formed by polyuridylic acid 3′,5′‐cyclic AMP

Abstract: The existence of a soluble complex formed by polyuridylic acid (poly (U)) and 3′,5′-cyclic AMP (cAMP) is demonstrated by u.v. extinction vs. temperature curves, optical rotation, equilibrium dialysis, and reaction calorimetry. The complex hasthe stoichiometry of 2 poly (U)-cAMP and its formation is accompanied by an enthalpy change of −13.0 kcal/mole of base triplet. The introuction of an empirical factor α in the equations given by Damle2 and Crothers2 leads to the evolution of a ΔH value of −13.4 keal/mole. The parameter α is considered as a correction factor for the concentration dependence of the binding process. There is no relation between α and the reduction of monomer activity due to self-association of monomers. The study of the binding process at several temperatures showed that the cooperativity parameter, σ, is independent of temperature and its value of 6.5 × 10−3 is in good agreement with σ = 5 × 10−3 for the poly (U)·poly(A) system.3

Conformation of Mixed Liganded Hybrids as Detected by their Interaction with Hb CHarlem

Abstract: THE sigmoid curve which represents the binding of oxygen by haemoglobin (Hb)1 has defied unequivocal explanation so far. Several models can account for many of the observed properties of Hb-ligand interaction2–4, but many questions exist concerning the details of the conformation changes in the haemoglobin tetramer which underlie the cooperativity displayed by the four haem groups. Any explanation of this behaviour must describe the conformation and properties of Hb molecules which are partially liganded. Several investigators have studied the behaviour of models of such intermediate ligand states, viz. artificially prepared mixed ligand hybrids5 in which either the α chains or the β chains are fixed in the liganded state while the partner chains can reversibly bind ligand and M haemoglobins6 in which the two haems of the mutant globin chains (α or β) are in the ferric state. While Perutz7 demonstrated the occurrence of two end states with specific quaternary conformation, the conformation of Hb in intermediate liganded states is not known.

Cooperativity of the electroplax membrane

Abstract: Considerable attention has been given to the molecular basis of ‘cooperativity’ in macromolecules (Hill, 1910; Adair, 1925; Monod, Wyman & Changeux, 1965; Koshland & Neet, 1968; Perutz, 1969; Edelstein, 1971) and in biological membranes (Changeux, 1969). Cooperativity is defined generally as a reaction of order greater than one, and, in biology, possibly the best known example of a cooperative reaction is the binding of oxygen to haemoglobin. The binding curve for this reaction is sigmoid, and sigmoid binding curves have proved often to be the first indication of a cooperative reaction.