Showing papers on "Cooperativity published in 1976"

Theoretical calculations of the helix-coil transition of DNA in the presence of large, cooperatively binding ligands.

Abstract: Theoretical calculations are conducted on the helix–coil transition of DNA, in the presence of large, cooperatively binding ligands modeled after the DNA-binding proteins of current biological interest. The ligands are allowed to bind both to helx and to coil, to cover up any number of bases or base pairs in the complex, and to interact cooperatively with their nearest neighbors. The DNA is treated in the infinite homogeneous Ising model approximation, and all calculations are done by Lifson's method of sequence-generating functions. DNA melting curves are calculated by computer in order to expolore the effects on the transition of ligand size, binding constant, free activity, and ligand–ligand cooperativity. The calculations indicate that (1) at the same intrinsic free energy change per base pair of the complexes, small ligands, for purely entropic reasons, are more effective than are large ligands in shifting the DNA melting temperature; (2) the response of the DNA melting temperature to increased ligand binding constant K and/or free ligand activity L is adequately represented at high values of KL (but not at low KL) by a simple independent site model; (3) if curves are calculated with the total amount of added ligand remaining constant and the free ligand activity allowed to vary throughout the transition, biphasic melting curves can be obtained in the complete absence of ligand–ligand cooperativity. In an Appendix, the denaturation of poly[d(A-T)] in the presence of the drug, netropsin, is used to verify some features of the theory and to illustrate how the theory can be used to obtain numerical estimates of the ligand binding parameters from the experimental melting curves.

Evidence for phase boundary lipid. Permeability of Tempo-choline into dimyristoylphosphatidylcholine vesicles at the phase transition.

Abstract: The existence of distinct regions of mismatch in molecular packing at the interfaces of the fluid and ordered domains during the phase transition of dimyristoylphosphatidylcholine vesicles has been demonstrated by measuring the temperature dependence of the permeability to a spin-label cation and comparing this with a statistical mechanical calculation of the fraction of interfacial lipid. The kinetics of uptake and release of the 2,2,6,6-tetramethylpiperidinyl-1-oxycholine (Tempo-choline) spin label by single-bilayer dimyristoylphosphatidylcholine vesicles were measured using electron spin resonance spectroscopy to quantitate the amount of spin label present within the vesicles after removal of the external spin-label by ascorbate at 0 degrees C. Both the uptake and release experiments show that the Tempo-choline permeability peaks to a sharp maximum at the lipid-phase transition, the vesicles being almost impermeable to Tempo-choline below the transition and having a much reduced permeability above. The temperature profile of the permeability is in reasonable quantitative agreement with calculations of the fraction of interfacial boundary lipid from the Zimm and Bragg theory of cooperative transitions, which use independent spin-label measurements of the degree of transition to determine the cooperativity parameter. The relatively high intrinsic permeability of the interfacial regions (P approximately 0.2-1.0 X 10(-8) cm/s) is attributed to the mismatch in molecular packing of the lipid molecules at the ordered-fluid boundaries, which could have important implications not only for permeability in natural membranes (e.g., in transmitter release), but also for the function of membrane-bound enzymes and transport proteins.

Cooperative properties of hormone receptors in cell membranes

Abstract: The binding of many polypeptide hormones to cell surface receptors does not appear to follow the law of mass action. While steady-state binding data are consistent in many cases with either heterogeneous populations of binding sites or interactions of the type known as negative cooperativity, study of the kinetics of dissociation of the type known as negative cooperativity, study of the kinetics of dissociation of the hormone receptor complex allows an unambiguous demonstration of cooperative interactions. Negative cooperativity, which seems to be wide-spread among hormone receptors, provides exquisite sensitivity of the cell at low hormone concentrations while buffering against acutely elevated hormone levels. The molecular mechanisms underlying the cooperativity are still largely unknown. Cooperativity may stem from a conformational transition in individual receptors or involve receptor aggregation in the fluid membrane (clustering) or more extensive membrane phenomena. Thus, new models of hormone action must be considered which integrate the progress in our knowledge of both the complex mechanisms regulating hormone binding to their surface receptors, and the dynamic properties of the cell membrane.

Oxygenation-linked subunit interactions in human hemoglobin: experimental studies on the concentration dependence of oxygenation curves.

Abstract: An experimental study on the concentration dependence of oxygenation curves for human hemoglobin has been carried out between 4 X 10(-8) M heme and 5 X 10(-4) M heme in 0.1 M tris(hydroxymehtyl)aminomethane hydrochloride, 0.1 M NaCl, 1 mM disodium ethylenediaminetetraacetic acid, pH 7.4, 21.5 degrees C. With decreasing hemoglobin concentration the curves show pronounced shifts in position and shape, consistent with dissociation of tetrameric hemoglobin into dimeric species of high affinity and low cooperativity. Combination of these data with independently determined values of dissociation constants for unliganded and fully liganded hemoglobin permits a resolution of the seven parameters necessary to define the linked binding and subunit association processes. The total oxygenation-linked subunit dissociation energy (6.34 kcal) was resolved into intersubunit contact energy changes between alphabeta dimers in tetrameric hemoglobin which accompany binding of the first, middle two, and last oxygen molecules. The resolution is accurate to within approximately +/-0.3 kcal. To within this limit the isolated dimers are found to bind oxygen noncooperatively and with the same affinity as isolated alpha and beta chains. Equally good fits to the data are obtained when dimers are slightly anticooperative. At least three major energetic states are apparently assumed by hemoglobin tetramers, with respect to the alpha1beta2 contact region, corresponding to (a) unliganded, (b) singly liganded, (c) triply and quadruply liganded species. The results do not establish whether these states may be assumed by a single molecule, or whether they arise as averages over a distrubution of conformational states. They do provide unequivocal evidence against a concerted transition at any particular binding step in a system with only two energetic states of tetramer (i.e., an all or none switchover between T and R states at a particular binding step).

The Role of Negative Cooperativity and Half-of-the-Sites Reactivity in Enzyme Regulation

Abstract: Publisher Summary This chapter discusses the role of negative cooperativity and half-of-the-sites reactivity in enzyme regulation. Negative cooperativity refers to the phenomenon in multisubunit proteins in which the binding affinities of ligands decrease as a function of ligand saturation. In positive cooperativity, the affinity of the protein toward the ligand increases as a function of ligand saturation, thus leading to sigmoidal saturation curves. The chapter analyzes the phenomenon of negative cooperativity from the theoretical point of view, discusses diagnostic tests for the property, and describes some well studied regulatory enzymes. The analysis of the binding of a single ligand to a multisubunit protein is in itself a fairly complex mathematical problem. The problem becomes Herculean when more than one ligand is involved, and yet that is precisely the case for regulatory proteins that usually have more than one substrate, more than one product, several regulatory molecules, and between two and eight subunits, each of which can combine with any of these molecules.

Transition from noncooperative to cooperative and selective binding of histone H1 to DNA.

Abstract: A transition from noncooperative to cooperative binding of DNA and histone h1 occurs between 20 and 40mMNaCI in 5 mM Tris-HCI, pH 7.5. Below 20mM NaCI in mixtures in H1 and excess DNA, H1 binds to all of the DNA molecules, causing them to sediment faster, and does not distinguish between DNA molecules that differ in size or base composition. However, at NaCI concentrations above the narrow transition range, H1 binds to only some of the DNA molecules and leaves the rest free. If the DNA molecules in a mixture are the same size, H1 selectively binds those that have the highest content of adenosine (A)+thymidine (T). By means of competion experiments at salt concentrations spanning the transition range, it is demonstrated that H1 selectivity requires cooperativity. A high degree of selectivity based on A + T content can be produced by cooperative binding: for average DNA sizes of 2 X10(6) daltons, more than ten molecules of calf lymphocyte DNA (57% a+ t) are chosen per molecule of Escherichia coli DNA (50% A+T).

Production of yeast alcohol dehydrogenase isoenzymes by selection.

Abstract: Mutants of yeast alcohol dehydrogenase have been produced that protect the cell against the poisonous aldehyde acrolein by increasing the NADH-NAD ratio. The altered properties include changes both in binding constants and in cooperativity. Such mutants may be useful in exploring the nature of adaptation at the molecular level.

(Na' , K+)-Activated Adenosinetriphosphatase of Axonal Membranes, Cooperativity and Control

Abstract: 1 The ATP sites Homotropic interactions between ATP sites have been studied in a very large range of Na+ and K+ concentrations The ( Na+, K+)-activated ATPase displays Michaelis-Menten kinetics for ATP under standard concentration conditions of Na+ (100 mM) and K+ (10 mM) The steady-state kinetics behavior changes at very low concentrations of K+ where negative cooperativity is observed The existence of a high affinity and a low affinity site for ATP was clearly demonstrated from the study of the ATP stimulated hydrolysis of p-nitrophenylphosphate in the presence of Na+ and K+ The ratio of apparent affinities of high and low affinity sites for ATP is 86 at pH 75 2 The Na+ sites The binding of Na+ to its specific stimulatory sites (internal sites) is characterized by positive cooperativity with a Hill coefficient n(H(Na+))=20 Homotropic interactions between Na+ sites are unaffected by variations of the K+ concentration 3 The K+ sites (a) Binding of K+ to the (external) stimulatory site of the ATPase has been analyzed by following the (Na+, K+)-ATPase activity as well as the p-nitrophenylphosphatase activity in the presence of Na+ and K+ (with or without ATP) Binding is characterized by a Hill coefficient of 10 and a K(05(K+))=01 to 08 mM The absence of positive or negative cooperativity persists between 5 mM and 100 mM Na+ (b) The analysis of the p-nitrophenylphosphatase or of the 2, 4 dinitrophenylphosphatase activity in the presence of K+ alone indicates the existence of low affinity sites for K+ with positive homotropic interactions The characteristics of stimulation in that case are, K(05)=5 mM, n(H)=19 The properties of this family of site(s) are the following: firstly, saturation of the low affinity site(s) by K+ prevents ATP binding to its high affinity internal site Secondly, saturation of the low affinity sites for K+ prevents binding of Na+ to its internal sites Thirdly, this family of sites disappears in the presence of ATP, p-nitrophenylphosphate or of both substrates, when Na+ binds to its internal sites Na+ binding to its specific stimulatory sites provokes the formation of the high affinity type of site for K+ 4 Mg2+ stimulation of the (Na+, K+)-ATPase is characterized by a Hill coefficient n(H(Mg2+))=10 and a K(05(Mg2+))=1 mM stimulation is essentially a V effect Heterotropic effects between binding of Mg2+ and substrate to their respective sites are small Heterotropic interactions between the Ms2+, Na+ and K+ sites are also small 5 The fluidity of membrane lipids also controls the (Na+, K+)-ATPase activity Phase transitions or separations in the membrane hardly affect recognition properties of substrates, Na+, K+ and Mg2+ for their respective sites on both sides of the membrane Only the rate of the catalytic transformation is affected

Purification and Properties of Cyclopentanone Oxygenase of Pseudomonas NCIB 9872

Abstract: 1 Cyclopentanone oxygenase from Pseudomonas NCIB 9872 has been purified some 40-fold. It gives a single peak in the ultracentrifuge and a single major protein band on polyacrylamide gels contaminated with about 5% of a slower migrating impurity. Flavin dissociates from the protein during electrophoresis. 2 The enzyme has a molecular weight of about 200000 and is a homopolymeric assemblage of either three or four subunits of molecular weight 54000–58000. 3 The prosthetic group is FAD and values of about 2.5 are typically obtained for the number of moles bound to each mole of holoenzyme. Some FAD probably dissociates during purification and it seems likely that each subunit binds one FAD in the undamaged protein. 4 The unitary stoichiometry of cyclopentanone, oxygen and NADPH is typical of mixedfunction oxygenases with external electron donors. The oxygenated product has been identified as 1-oxa-2-oxocyclohexane (5-valerolactone) and enzyme is therefore systematically named as cyclopentanone, NADPH: oxygen oxidoreductase (1,2-lactonizing) (EC 1.14.13.–). 5 Catalytically functional sulfhydryl groups are probably present but the biphasic nature of the inhibition curve when enzyme is titrated with p-hydroxymercuribenzoate reveals the presence of six titratable groups that are not all equivalent. 6 The enzyme has a pH optimum of 7.7. The Km for cyclopentanone is below 1 μM but not experimentally determinable. The response to variation of NADPH concentration is sigmoidal, indicative of homotrophic binding, a Hill plot gave an interaction number of 3, perhaps indicative of cooperativity between three subunits. 7 Anaerobic reduction of enzyme-bound FAD by addition of NADPH required only slightly in excess of two electron equivalents. No evidence for the formation of stable semiquinones was obtained in this or in photoreduction experiments. 8 Though the enzyme displays sensitivity towards transition metal chelating agents the small amounts of Fe and Cu present make a role in electron transport unlikely. The possibility that they may be involved in maintenance of quaternary structure has not been investigated.

Evidence for chromophore-chromophore interactions in the Purple Membrane from reconstitution experiments of the chromophore-free membrane

Abstract: We recently presented evidence showing that the visible CD spectrum of the purple membrane from Halobacterium halobium consists of two contributions: a broad positive band centered at the absorption maximum due to the interaction of the chromophore with the protein to which it is bound, and an exciton coupling band due to the interaction between chromophores of adjacent bacteriorhodopsin molecules in the hexagonal surface lattice (Heyn et al., 1975). This interpretation receives strong support from the present experiments in which the chromophore-free membrane is reconstituted by the addition of retinal. Since the coupling signal arises from the interaction between pairs of neighboring chromophores, its contribution to the spectrum would be expected to be very small in the initial stages of the titration experiment, but increasing quadratically with the percentage reconstitution. The broad positive band, on the other hand, is expected to increase linearly with the percentage reconstitution. On the basis of these considerations a satisfactory explanation of the CD reconstitution experiments could be given. Since it appears to be impossible to explain the titration experiments without the quadratic term, we conclude that chromophore-chromophore interactions play an important role. No significant changes in secondary structure upon reconstitution could be detected consistent with our binding model which neglects cooperativity.

Evidence for negative cooperativity in the adsorption of phosphorylase b on hydrophobic agaroses.

Abstract: The concept of cooperativity appears to be the key to the understanding of the complex mechanisms underlying the adsorption of proteins to agaroses substituted with hydrophobic alpha-aminoalkanes. The adsorption of phosphorylase b occurs through the positive cooperative interaction of a critical number of approximately 3-5 butyl and a higher number of methyl residues with ocrresponding sites on the enzyme. The amount of adsorbed phosphorylase b per millileter of packed gel (methyl-, butyl-Sepharose) in the absence and presence of 1.1 M ammonium sulfate at temperatures between 0 and 34 degrees C is a power function of the free solute equilibrium concentration (Freundlich isotherm). In contrast, the adsorption of cyanmyoglobin to phosphocellulose is described by the Langmuir equation. The surface coverage dependent isosteric heats of adsorption for phosphorylase b indicate an endothermic reaction only on the butyl-Sepharose in the presence of high salt concentrations. Scatchard plots of the Freundlich isotherms of phosphorylase b are concave upwards, typical of negative cooperativity. Hill plots of these isotherms (5-70% saturation) yield coefficients between nH = 0.39 and 0.71. At high surface coverages, the Hill coefficients approach unity. Apparent association constants (K0.5) of 4-39 X 10(4) M-1 are calculated for the adsorption of phosphorylase b, as compared to 2-9 X 10(4) M-1 for the adsorption of cyanmyoblobin. In general, negative cooperativity of binding may be explained by changes in the affinity of the ligand for the matrix, due to the sequential multivalent adsorption, and competition of phosphorylase b molecules for the critical number of alkyl residues (nonindependence of binding) on one side and to variations in the configuration of binding and entropy on the other.

On present theories of the condensed polymer state

Abstract: An improvement of the bundle model gives the possibility to discuss quantitatively the solid state transition and the final melting of PE and calculate the transition data as well as the cooperativity. A similar quantitative treatment is carried out to explain the kink-block transitions in clay organic complexes.

Hemoglobin McKees Rocks (alpha2beta2145Tyr leads to Term). A human "nonsense" mutation leading to a shortened beta-chain.

Abstract: The first example of the premature termination of a polypeptide chain in man appears to be Hb McKees Rocks, beta145 Tyr leads to Term, discovered in polycythemic members of a Caucasian family. Point mutation has apparently occurred at the codon for Tyr beta145 from UAU to a "nonsense" codon, UAA or UAG, resulting in a shortened polypeptide chain with Lys 144 as its carboxyl-terminal amino acid. Evidence for this structural conclusion is the absence of tryptic peptide betaT-15 from "fingerprints" of the abnormal beta-chain, the finding of C-terminal Lys, and the similarity between the functional properties of this variant hemoglobin and those of des Tyr (145)-His(146)beta hemoglobin resulting from carboxypeptidase-A digestion of normal human hemoglobin. Hb McKees Rocks has markedly abnormal properties: its oxygen affinity is the highest of the human variants described to date; its Bohr effect is reduced; it is devoid of subunit cooperativity; and it is unaffected by 2,3-diphosphoglyceric acid. These properties are probably the consequences of decreased stability of the T quaternary conformation and are partially restored in the presence of the strong allosteric effector inositol hexaphosphate.

The Oxygenation of Hemoglobin in Lugworms

Abstract: The extracellular hemoglobin found in the closed circulatory system of lugworms has a high oxygen affinity and extremely great cooperativity in oxygen binding. These properties are not accompanied by low oxygen levels in the microhabitat so long as ventilation continues. The quantitative function of lugworm hemoglobin is greatest at the intermediate and high oxygen levels created in the burrow by maximum ventilation activity. The distinctive oxygenation properties are related to operating conditions in the blood vessels. The large amount of mixing of post- and prebranchial blood in this primitive circulatory system results in low blood PO₂'s and little PO₂ difference between afferent and efferent blood. The Bohr shift is functional, but its role in oxygen transport is small. The primary function of lugworm hemoglobin is oxygen transport at high tide. At both low and high acclimation temperatures, an appreciable fraction of the oxygen consumed (15%-45%) is carried by hemoglobin.

Alteration of the allosteric properties of aspartate transcarbamoylase by pyridoxylation of the catalytic and regulatory subunits.

Abstract: Extension modification of aspartate transcarbamoylase from Escherichia coli with pyridoxal 5'-phosphate followed by reduction of the Schiff base with sodium borohydride caused only partial inactivation of the enzyme. Under comparable conditions, virtually complete loss of enzyme activity is obtained with the free catalytic subunits. The pyridoxylated, intact enzyme containing more than 60% of the bound pyridoxamine phosphate on the regulatory subunits exhibited considerable cooperativity, inhibition by CTP, and activation by ATP. When the modification was performed in the presence of the ligands which bind to the catalytic sites, the resulting product had virtually the same activity as the native enzyme, but it exhibited significantly reduced cooperativity and virtually no inhibition by CTP. The pyridoxylation of the regulatory subunits within the intact enzyme was enhanced markedly in the presence of ligands as compared with the reactivity of these subunits when the modificaiton was performed in the absence of the active site ligands. Both types of pyridoxylated derivatives exhibited the ligand-promoted conformational changes characteristic of the native enzyme. Spectrophotometric studies of inactive pyridoxylated catalytic subunits and intact enzyme showed that the substrate (carbamoyl phosphate) bound strongly but that the substrate analogue (succinate) did not bind. Both the pyridoxylation experiments in the presence and absence of ligands and the spectral behavior of a hybrid containing one native and one pyridoxylated catalytic subunit indicated that ligand binding was accompanied by a conformational change in the intact enzyme molecules.

Altered C-terminal salt bridges in haemoglobin york cause high Oxygen affinity

Abstract: STUDIES of abnormal human haemoglobins (Hbs) with increased O2 affinity have been particularly useful in developing detailed molecular interpretations of normal Hb function. A number of functionally interesting mutants are altered in the βHC region but only one variant, Hb Hiroshima (β146 His→Asp)1,2, has been reported as having a substitution at the C terminus itself. We report here the structure and O2 equilibria of Hb York, a new mutation at the β146 position, His→Pro, associated with increased O2 affinity, decreased cooperativity and diminished Bohr effect.

Alkylation of estradiol 17beta-dehydrogenase from human placenta with 3-chloroacetylpyridine--adenine dinucleotide.

Abstract: 3-Chloroacetylpyridine–adenine dinucleotide phosphate is both active as a hydride acceptor and inactivates estradiol 17β-dehydrogenase. This coenzyme analogue behaves like an affinity label. The inactivation kinetics are discussed in relation to those observed with 3-chloroacetylpyridine–adenine dinucleotide. The pH dependence of the rate of inactivation, in combination with determination of the number of reactive cysteine residues, pointed to the alkylation of one cysteine residue/subunit. The stoichiometry was one molecule of dinucleotide per subunit and no cooperativity was detected. When 14C-labeled dinucleotide was used, the 14C label was found mainly in one peptide, accounting for 90% of the incorporated radioactivity, whereas in previous work it had been shown that 3-chloroacetylpyridine–adenine dinucleotide is an affinity reagent which labels three peptides.

Ligand interactions with the acetylcholine receptor from Torpedo californica. Extensions of the allosteric model for cooperativity to half-of-site activity.

Abstract: The solubilized acetylcholine receptor from Torpedo californica showed positive cooperativity in acetylcholine binding with a dissociation constant of 1.2 X 10(-8) M. Blockade of acetylcholine binding by nicotine was competitive; blockade by d-tubocurarine appeared to result from an allosteric interaction that altered half of the acetylcholine binding sites to a lower affinity form; decamethonium blockade displayed properties of competitive and allosteric inhibition suggesting less specificity for decamethonium binding than seen with either nicotine or d-tubocurarine. The d-tubocurarine inhibition data were evaluated by several possible models involving either differential competitive inhibition or allosteric inhibiton. The data were best described by the allosteric model.