Showing papers on "Cooperative binding published in 1985"

Calcium binding to complexes of calmodulin and calmodulin binding proteins.

Abstract: The free energy of coupling for binding of Ca2+ and the calmodulin-sensitive phosphodiesterase to calmodulin was determined and compared to coupling energies for two other calmodulin binding proteins, troponin I and myosin light chain kinase. Free energies of coupling were determined by quantitating binding of Ca2+ to calmodulin complexed to calmodulin binding proteins with Quin 2 to monitor free Ca2+ concentrations. The geometric means of the dissociation constants (-Kd) for Ca2+ binding to calmodulin in the presence of equimolar rabbit skeletal muscle troponin I, rabbit skeletal muscle myosin light chain kinase, and bovine heart calmodulin sensitive phosphodiesterase were 2.1, 1.1, and 0.55 microM. The free-energy couplings for the binding of four Ca2+ and these proteins to calmodulin were -4.48, -6.00, and -7.64 kcal, respectively. The Ca2+-independent Kd for binding of the phosphodiesterase to calmodulin was estimated at 80 mM, indicating that complexes between calmodulin and this enzyme would not exist within the cell under low Ca2+ conditions. The large free-energy coupling values reflect the increase in Ca2+ affinity of calmodulin when it is complexed to calmodulin binding proteins and define the apparent positive cooperativity for Ca2+ binding expected for each system. These data suggest that in vitro differences in free-energy coupling for various calmodulin-regulated enzymes may lead to differing Ca2+ sensitivities of the enzymes.

cGMP-dependent protein kinase: autophosphorylation changes the characteristics of binding site 1

Abstract: cGMP-dependent protein kinase binds 4 mol cGMP/mol enzyme to two different sites. Binding to site 1 (apparent Kd 17 nM) shows positive cooperativity and is inhibited by Mg . ATP, whereas binding to site 2 (apparent Kd 100-150 nM) is non-cooperative and not affected by Mg . ATP. Autophosphorylation of the enzyme abolishes the cooperative binding to site 1 and the inhibitory effect of Mg . ATP. The association (K1) and dissociation (K-1) rate constant for site 2 and K1 for site 1 are not affected significantly by Mg . ATP or autophosphorylation. The dissociation rate from site 1 measured in the presence of 1 mM unlabelled cGMP is decreased threefold and over tenfold by Mg . ATP and autophosphorylation, respectively. In contrast, the dissociation rate from site 1 measured after a 500-fold dilution of the enzyme-ligand complex is 100-fold faster than that determined in the presence of 1 mM cGMP and is only slightly influenced by Mg . ATP or autophosphorylation. Only Kd values calculated with the latter K-1 values are similar to the Kd values obtained by equilibrium binding. These results suggest that autophosphorylation of cGMP-dependent protein kinase affects mainly the binding characteristics of site 1.

Interaction of opiates with opioid binding sites in the bovine adrenal medulla: I. Interaction with delta and mu sites.

Abstract: In the present study we examined the interaction of opiates with the delta and mu opioid binding sites in the bovine adrenal medulla. [3H][D-Ala2, D-Leu5]-enkephalin ( [3H]DADLE) in the presence of saturating concentrations of morphiceptin was used to analyze delta site interactions, whereas either [3H]DADLE in the presence of saturation concentrations of [D-Ser2, Leu5]-enkephalin-Thr6 (DSLET) or [3H][D-Ala2, Me-Phe4, Gly5-ol]-enkephalin ( [3H]DAGO) was used for the determination of mu sites. Both binding sites were found to interact stereoselectively with opiates. The binding was affected differentially by proteolytic enzymes (trypsin, alpha-chymotrypsin, pepsin), N-ethylmaleimide, and A2-phospholipase. Kinetic and equilibrium binding studies revealed that in each case radiolabeled opiates interact with one class of binding sites, following simple second-order bimolecular kinetics. Competition for binding by opiates and opioid peptides confirmed the delta and mu selectivity of these sites. Monovalent (Na+, Li+, K+) and divalent (Mg2+, Mn2+, Ca2+) ions interacted differentially with these two binding sites: In general, monovalent cations affected preferentially the apparent number of binding sites, whereas divalent ions modified the equilibrium dissociation constant. Furthermore, positive or negative cooperativity and an apparent heterogeneity of binding sites were detected under some ionic conditions.

Virus-receptor interaction in the adenovirus system: characterization of the positive cooperative binding of virions on HeLa cells.

Abstract: The established positive cooperativity of adenovirus 2 binding to HeLa cells revealed a strong temperature dependence. The degree of cooperativity, quantified by means of Hill coefficients, progressively increased from 10 degrees C to reach a maximum level, which was maintained between 20 and 37 degrees C. On the other hand, negative cooperativity of virion attachment was apparent at 3.0 degrees C and on glutaraldehyde-stabilized cells. The corresponding monovalent ligand of the system, the fiber antigen, demonstrated only weak-positive cooperativity of the binding at 37.0 degrees C, which was absent at 3.0 degrees C. Dithiothreitol and dansylcadaverine, reagents inhibiting clustering of ligand-receptor complexes in the plasma membrane, markedly reduced the degree of positive cooperative binding at 37.0 degrees C. Evidently, the positive cooperative binding of adenovirus to HeLa cells at 37.0 degrees C is a consequence of both the multivalency of virus attachment proteins, i.e., fibers, on the virion and of the capacity of the receptor sites to migrate in the plane of the plasma membrane, forming local aggregates of virus-receptor site complexes.

Adhesion of Pseudomonas aeruginosa to human buccal epithelial cells: evidence for two classes of receptors.

Abstract: The adhesion of Pseudomonas aeruginosa strain 492c to trypsinized and untrypsinized buccal epithelial cells (BECs) was studied. Kinetic analysis of the adhesion data, employing a Langmuir absorption isotherm, indicated the presence of two classes of binding sites on untrypsinized BECs: a high affinity – low copy number site (apparent association constant (Ka ≈ 1.57 × 10−8 mL/cell with ca. 29 binding sites/cell) and a low affinity – high copy number class of binding sites (Ka ≈ 4.78 × 10−10 mL/cell with ca. 264 binding sites/cell). The low affinity – high copy number class of sites was found to be trypsin sensitive. A single class of binding sites was found on trypsinized BECs exhibiting a high affinity – low copy number (Ka ≈ 3.70 × 10−7 mL/cell with ca. 31 binding sites/cell). Positive cooperativity in binding of P. aeruginosa strain 492c to the low affinity – high copy number class site on untrypsinized BECs was demonstrated by analysis of Hill plots of the adhesion data. Sugar inhibition data using a p...

Reversible dissociation of dimeric tyrosyl-tRNA synthetase by mutagenesis at the subunit interface.

Abstract: Dimeric tyrosyl-tRNA synthetase from Bacillus stearothermophilus exhibits half-of-the-sites reactivity and negative cooperativity in binding of tyrosine. Protein engineering has been applied to the enzyme to determine whether it can be reversibly dissociated into monomers and if the monomers are active. The target for mutation is the residue Phe-164. The side chain of Phe-164 in one subunit interacts with its symmetry-related partner in the other. Mutation of Phe-164----Asp-164 gives a mutant [TyrTS(Asp-164)] that undergoes dissociation at high pH when the aspartate residues are ionized. The monomer is inactive and does not bind tyrosine. Dissociation is enhanced at low concentrations of enzyme by a mass action effect. Kinetic and binding measurements on TyrTS(Asp-164) with tyrosine and tyrosyl adenylate show that the monomer has very weak affinity for these ligands. Accordingly, dimerization is favored by high concentrations of tyrosine and ATP since the dimeric form has a high affinity for the ligands. The presence of tRNA does not encourage dimer formation, and so it must bind to the monomer. TyrTS(Asp-164) is fully active at pH 6 where dimerization is favored but has low activity at pH 7.8 where dissociation is favored. It should now prove possible to engineer heterodimers that may be used to investigate the subunit interactions further.

Long-range allosteric effects on the B to Z equilibrium by daunomycin

Abstract: Spectroscopic and fluorometric methods were used to study the binding of the anticancer drug daunomycin to poly[d(G-C)] and poly[d(G-m5C)] under a variety of solution conditions. Under high-salt conditions that favor the left-handed Z conformation, binding isotherms for the interaction of the drug with poly[d(G-C)] are sigmoidal, indicative of a cooperative binding process. Both the onset and extent of the cooperative binding are strongly dependent upon the ionic strength. The binding data may be explained by a model in which the drug preferentially binds to B-form DNA and acts as an allosteric effector on the B to Z equilibrium. At 2.4 M NaCl, binding of as little as one drug molecule per 20 base pairs (bp) results in the conversion of poly[d(G-C)] from the Z form entirely to the B form, as inferred from binding data and demonstrated directly by circular dichroism measurements. Similar results are obtained for poly[d(G-m5C)] in 50 mM NaCl and 1.25 mM MgCl2. Under these solution conditions, it is possible to demonstrate the Z to B structural transition in poly[d(G-m5C)] as a function of bound drug by the additional methods of sedimentation velocity and susceptibility to DNase I digestion. The transmission of allosteric effects over 20 bp is well beyond the range of the drug's binding site of 3 bp. Since daunomycin preferentially binds to alternating purine-pyrimidine sequences, which are the only sequences capable of the B to Z transition, the allosteric effects described here may be of importance toward understanding the mechanism by which the drug inhibits DNA replicative events.(ABSTRACT TRUNCATED AT 250 WORDS)

Mechanism of allosteric regulation of the Ca,Mg-ATPase of sarcoplasmic reticulum: studies with 5'-adenylyl methylenediphosphate.

Abstract: Four mechanisms for the allosteric regulation of the calcium and magnesium ion activated adenosinetriphosphatase (Ca,Mg-ATPase) of sarcoplasmic reticulum were examined. Negative cooperativity in substrate binding was not supported by 3H-labeled 5'-adenylyl methylenediphosphate (AMPPCP) binding, which was best fit by a single class of sites. Although calcium had no effect on the absence of cooperativity, it did increase the affinity of the enzyme for AMPPCP. Allosteric regulation via an effector site for AMPPCP or ATP on the same ATPase chain was eliminated by the stoichiometry of ATP and AMPPCP binding, 1 mol of site per mole of enzyme. The possibility that AMPPCP acts at an effector site was eliminated by showing that it competitively inhibits the rate of phosphoenzyme formation. Allosteric regulation of kinetics via site-site interaction in an oligomer was eliminated by showing that the inhibition of ATPase activity by fluorescein isothiocyanate is linearly dependent upon its incorporation into the sarcoplasmic reticulum. The fourth mechanism considered was stimulation of ATPase activity by the binding of ATP or AMPPCP at the active site after departure of ADP but before the departure of inorganic phosphate. This hypothesis was supported by site stoichiometry and by the observation that AMPPCP or ATP stimulates v/EP, the rate of ATP hydrolysis for a given level of phosphoenzyme. Computer simulation of this branched monomeric model could duplicate all experimental observations made with AMPPCP and ATP as allosteric regulators. The condition that the affinity of ATP binding to the enzyme be reduced when it is phosphorylated, which is required by the computer model, was confirmed experimentally.

Oxygen equilibria of Octopus dofleini hemocyanin

Abstract: Oxygen binding by Octopus dofleini hemocyanin was examined under very nearly physiological conditions. The effects of pH, ionic composition, temperature, and aggregation were controlled so that the role each plays in modulating oxygen binding can be isolated. There is a very large effect of pH on affinity, the Bohr effect (delta log P50/delta pH = -1.7), which is the same at 10 and 20 degrees C. However, cooperativity is substantially altered over the same range of pHs at the two temperatures. The allosteric properties were examined by comparing the experimental data points to curves generated by use of the Monod-Wyman-Changeux model. A computer-fitting process was developed which allowed the individual allosteric parameters to be varied independently until the best fit could be determined. The relationship between kR and kT is responsible for the effect of pH on cooperativity. A change in the allosteric properties of the T form is primarily responsible for the differences due to temperature. Changing cation concentrations when the molecule is in the fully aggregated 51S form alters affinity without influencing cooperativity. The effect of Mg2+ is much greater than that of Na+. If the 51S decamer is dissociated to 11S monomers by removing divalent cations, oxygen binding is noncooperative. There is evidence for negative cooperativity, indicating heterogeneity of function within the subunit which contains seven oxygen binding domains. Association into decamers generates conformational change which results in a much wider range of allosteric function.

Binding of oxygen and carbon monoxide to arthropod hemocyanin: an allosteric analysis.

Abstract: The binding of oxygen and carbon monoxide to hemocyanin from the mangrove crab Scylla serrata and the lobster Homarus americanus has been studied by thin-layer optical absorption and front face fluorescence techniques. Three types of experiments were performed on subunit and oligomeric preparations of each hemocyanin: oxygen binding, carbon monoxide binding, and oxygen-carbon monoxide competition studies. The results obtained from the subunit preparations of dissociated oligomers from both hemocyanins show that the binding site can be ligated by either one oxygen or one carbon monoxide. The binding results obtained with the oligomeric samples of hemocyanin from both species cannot be described by the two-state MWC model [Monod, J., Wyman, J., & Changeux, J. P. (1965) J. Mol. Biol. 12, 88-118] since the data from the three types of binding experiments cannot be fit with a single set of binding constants. The MWC model has been extended by including a third allosteric form, and an analysis based on the three-state model is able to fit the data from the three types of experiments with the same set of binding constants. The comparison of the oxygen to carbon monoxide affinity ratios (kO2/kCO) indicates that the structure around the binding site of subunits in the T form oligomer is similar to that of the free subunits. The oligomeric forms of both these hemocyanins bind carbon monoxide with a weak but definite positive cooperativity. An analysis of the affinity ratios for the T, S, and R forms suggests that the high affinity of the R form results from a specific interaction between oxygen and binding site.

Covalent modification of both cAMP binding sites in cAMP-dependent protein kinase I by 8-azidoadenosine 3',5'-monophosphate.

Abstract: Reconstituted porcine cAMP-dependent protein kinase type I was labeled with 8-azidoadenosine 3',5'-monophosphate (8-N3cAMP) to study cyclic nucleotide binding and to identify amino acid residues that are either in or in close proximity to the cAMP binding sites. The photoaffinity analogue 8-N3cAMP behaved as cAMP itself with respect to cyclic nucleotide binding. For both cAMP and 8-N3cAMP, 2 mol of nucleotide was bound per mole of type I regulatory subunit monomer (RI), the apparent Kd's observed were approximately 10-17 nM on the basis of either Millipore filtration assays, equilibrium dialysis, or ammonium sulfate precipitation, Scatchard plots showed positive cooperativity, and (4) the Hill coefficients were approximately 1.5-1.6. After photolysis and addition of an excess of cAMP, approximately 1 mol of 8-N3cAMP/mol of RI monomer was covalently incorporated. Tryptic digestion of the labeled protein revealed that two unique tryptic peptides were modified. Proline-271 and tyrosine-371 were identified as the two residues that were covalently modified by 8-N3cAMP in RI. These results contrast with the type II regulatory subunit (RII) where 8-N3cAMP modified covalently a single tyrosine residue [Kerlavage, A. R., & Taylor, S. S. (1980) J. Biol. Chem. 255, 8483-8488]. RI contains two adjacent regions of sequence homology in the COOH-terminal fragment that binds two molecules of cAMP.(ABSTRACT TRUNCATED AT 250 WORDS)

Thermodynamic and kinetic cooperativity in ligand binding to multiple sites on a protein: Ca2+ activation of an ATP-driven Ca pump.

Abstract: Contrary to common belief, theoretical analysis does not predict any necessary relationship between cooperativity in the equilibrium binding of an ion to multiple binding sites on a protein and cooperativity in the kinetic activation of a reaction for which such binding is prerequisite. The sarcoplasmic reticulum Ca pump protein, for example, has two high-affinity binding sites for Ca2+, here considered to be nearly identical and independent. Equilibrium binding to these sites can be highly cooperative in spite of site-independence, as demonstrated by the well-known allosteric mechanism based on Wyman's principle of linked functions. We show in this paper that kinetic activation of the pump reaction cycle by binding of Ca2+ to these same sites can likewise be a cooperative function of Ca2+ concentration but that the criteria that determine cooperativity in the two situations are different. It is possible to observe kinetic cooperativity without concomitant cooperativity in equilibrium binding and vice versa. Application of these theoretical considerations to experimental data for the pump protein raises questions about the Ca2+ binding mechanism.

Properties of sodium-independent taurine binding to brain synaptic membranes.

Abstract: The sodium-independent taurine binding to twice Triton treated brain membranes characterized here was reversible, temperature-insensitive and saturable with a broad pH optimum, being thus characteristic of a synaptic receptor site. Only one type of binding site with a rather low maximal capacity was detected. Furthermore, binding exhibited properties of positive cooperativity, indicating that two or more taurine molecules possibly interact at a binding site. Both GABA and glycine as well as their agonists and antagonists strongly displaced taurine binding. The novel anticonvulsive taurine derivatives wee also effective displacers. The relevance of the binding observed in vitro to the possible synaptic receptors for taurine in vivo still remains to be determined.

Characterization of an insulin receptor in human Y79 retinoblastoma cells.

Abstract: Cultured human Y79 retinoblastoma cells bind [125I]iodoinsulin in a manner similar to that of other CNS and peripheral tissues. The only difference noted between the insulin binding properties of the Y79 cells and other CNS preparations is that insulin binding to Y79 cells is down-regulated by prolonged exposure of the cells to insulin. By contrast, studies with the various brain preparations indicate that the brain insulin receptor is not down-regulated by circulating levels of insulin. Insulin binding to Y79 cells exhibits negative cooperativity, has a pH optimum of 7.8, is responsive to cations, and gives a curvilinear Scatchard plot. Y79 cell insulin binding capacity is 26 fmol/100 micrograms of cell protein, corresponding to about 125,000 binding sites per cell. These findings are the first to report insulin binding in a human cell line of retinal origin. The characterization of the insulin binding in this cell line may facilitate an understanding of the relationship between insulin and its specific functions in the human retina.