Showing papers on "Cooperative binding published in 1980"
TL;DR: As expected, on the basis of the strong affinity of the enzyme for the calmodulin x Ca2+ complex, the Ca2- dependence of phosphodiesterase activation is highly cooperative and leads to a sharp threshold ofCa2+ concentration for control of enzyme activity.
Abstract: Equilibrium dialysis measurements of the binding of Ca2+ to calmodulin have confirmed the existence of four high affinity Ca2+-binding sites (Kd between 3 X 10(-6) and 2 X 10(-5) M). In the presence of 3 mM Mg2+, the dissociation constants for Ca2+ are increased two- to fourfold (Kd between 5 X 10(-6) and 4 X 10(-5) M). Positive cooperativity of Ca2+ binding was observed at low Ca2+ concentrations with Hill coefficients of 1.33 and 1.22 in the absence and presence of 3 mM Mg2+, respectively. The positive cooperativity is compatible with the steepness of the Ca2+ dependence of the conformational transition associated with the binding of 2 mol of Ca2+/mol of calmodulin. This conformational change, which affects the environment of the aromatic residues of calmodulin as measured by UV absorption and near-UV circular dichroism spectroscopy, is not the result of a monomer-dimer equilibrium mediated by Ca2+. Binding of Ca2+ to calmodulin is believed to occur by a sequential mechanism generating at least four different conformers of the protein and its free and liganded states. Even though the major conformational change is almost complete upon binding of 2 mol of Ca2+/mol of calmodulin, the activation of cyclic nucleotide phosphodiesterase measured in the presence of limiting concentrations of calmodulin suggests that a calmodulin Ca3-42+ complex is required for interaction of calmodulin with the enzyme. As expected, on the basis of the strong affinity of the enzyme for the calmodulin x Ca2+ complex (Kd = 1-3 X 10(-9) M), the Ca2+ dependence of phosphodiesterase activation is highly cooperative and leads to a sharp threshold of Ca2+ concentration for control of enzyme activity.
327 citations
TL;DR: Recent experimental data on the equilibrium binding of myosin subfragment 1 (S-1) to regulated actin filaments in the presence and in the absence of Ca(2+) are analyzed by using a linear Ising model.
Abstract: Recent experimental data on the equilibrium binding of myosin subfragment 1 (S-1) to regulated actin filaments in the presence and in the absence of Ca(2+) are analyzed by using a linear Ising model. In the model, each tropomyosin-troponin unit (including seven sites on the actin filament) can be in one of two possible states, which have different intrinsic free energies and different binding constants for S-1. Bound S-1 molecules do not interact with each other. There are nearest-neighbor (pair) interactions between these units that depend on the state of each member of the pair and on the number of Ca(2+) bound to one member of the pair. There are two sources of positive cooperativity in this system: the fact that seven actin sites change state together as part of a single unit; and the existence of attractive nearest-neighbor interactions between units. Parameters in the model are evaluated by fitting the data, both in the presence and in the absence of Ca(2+). Several extensions of this model are discussed.
257 citations
TL;DR: Results are consistent with a model that is basically similar to the cooperative binding model of Hill, where ADP remains weak until a higher free S-1 .
Abstract: The binding of myosin subfragment-1 (S-1) to the F-actin-troponin-tropomyosin complex (regulated F-actin) was examined in the presence of ADP (ionic strength, 0.23 M; 22 degrees C) by using the ultracentrifuge and S-1 blocked at SH1 with iodo[14C]acetamide. S-1 . ADP binds with positive cooperativity to regulated F-actin, both in the presence and absence of calcium; it binds independently to unregulated actin. With and without Ca2+ at very low levels of occupancy of the regulated actin by S-1 . ADP, S-1 . ADP binds to the regulated actin with less than 1% of the strength that it binds to unregulated actin, whereas at high levels of occupancy of the regulated actin by S-1 . ADP, S-1 . ADP binds about 3-fold more strongly to the regulated actin than it does to unregulated actin. The major difference between the results obtained in the presence and absence of Ca2+ with regulated actin is that, in the absence of Ca2+, the binding of S-1 . ADP remains weak until a higher free S-1 . ADP concentration is reached and the transition to strong binding is much more cooperative. These results are consistent with a model that is basically similar to the cooperative binding model of Hill[Hill, T.L. (1952) J. Chem. Phys. 20, 1259-1273] and of Monod et al. [Monod, J., Wyman, J. & Changeux, J. (1965) J. Mol. Biol. 12, 88-118]: The regulated actin filament can exist in two forms, a weak-binding and a strong-binding form; and Ca2+ and S-1 . ADP, acting as allosteric effectors, shift the equilibrium between the two forms.
244 citations
TL;DR: Hill plots of equilibrium dialysis data suggest that metal binding to cell walls involves negative cooperativity, and competition between various metals for binding sites suggested that the cations complex with identical sites on the cell walls.
Abstract: The use of equilibrium dialysis techniques established that isolated cell walls of Bacillus subtilis possess selective affinities for several cations. The binding of these cations to the cell wall was influenced by the presence of various functional groups in the peptidoglycan matrix. Selective chemical modification of the free carboxyl and amino groups showed that when amino groups were replaced by neutral, bulky, or negatively charged groups, the sites available for cation complexing generally increased. Introduction of positive charges into the wall resulted in a marked decrease in the numbers of metal binding sites and usually a decrease in the apparent association constants. Both teichoic acid and peptidoglycan contribute to the sites available for interaction with metals. Hill plots of equilibrium dialysis data suggest that metal binding to cell walls involves negative cooperativity. Competition between various metals for binding sites suggested that the cations complex with identical sites on the cell walls. When the hydrogen ion concentration was increased, the affinity of the walls for metals decreased, but the numbers of metal binding sites remained constant, suggesting that cations and protons also compete for the same sites.
242 citations
TL;DR: The transient-state kinetics of binding of myosin subfragment 1 (SF-1) to regulated actin in the presence and absence of Ca2+ were investigated and showed that partial occupancy of a block of G-actin sites by SF-1 or SF- 1-ADP stabilizes the open (active) conformation.
Abstract: The transient-state kinetics of binding of myosin subfragment 1 (SF-1) to regulated actin in the presence and absence of Ca2+ were investigated. The binding of SF-1 to pure actin, to actin-tropomyosin (actin-TM), or to actin-tropomyosin-troponin (actin-TM-TN) in the presence of Ca2+ was kinetically the same. In each case, the light-scattering transients were biphasic, suggesting a two-step binding of SF-1 to actin. Binding of SF-1 to regulated actin in the absence of Ca2+ was different from binding in its presence and also varied depending on whether SF-1 or regulated actin was in excess. The kinetic results in the absence of CA2+ are explained by a cooperative binding model, in which the initial binding of SF-1 molecules to open (active) actin sites increases the number of open sites. TN-I labeled with the fluorophore 4-(N-iodoacetoxyethyl-N-methyl)-7-nitrobenz-2-oxa-1,3 diazole (TN*) was used to probe the state of the actin-TM-TN complex. Binding of SF-1 or CA2+ to regulated actin (in the absence of Ca2+) decreased the fluorescence of actin-TM-TN* by 30%, suggesting that binding of SF-1 or CA2+ induces a similar change in state. The change in fluorescence of TN* was also used to measure the rate of the transition from the active to the relaxed state in the absence of CA2+, which was 430 sec-1 at 4 degrees C in 0.1 M KCl. The lag prior to association of SF-1 with regulated actin (in the absence of Ca2+) was abolished when three SF-1 molecules were prebound per seven G-actin monomers. Similarly, a titration of actin-TM-TN* (in the absence of Ca2+) with SF-1 or SF-1-ADP showed that most actin sites are open, as measured by the fluorescence change, when the occupancy of actin-TM-TN* by SF-1-ADP or SF-1 is approximately 50%. The evidence shows that partial occupancy of a block of G-actin sites (possibly seven) by SF-1 or SF-1-ADP stabilizes the open (active) conformation.
150 citations
TL;DR: The concept of the modification of enzyme activity by the allosteric binding of nucleotides, which is well established for soluble enzyme systems, also has application to the regulation of channels that control membrane permeability.
Abstract: The control by nucleotides of the Ca2+ -activated channel which regulates the nonspecific permeability of the mitochondrial inner membrane has been investigated quantitatively. The cooperative binding of two molecules of ADP to the internal (matrix) side of the channel causes a mixed-type inhibition of channel activity. ATP, AMP, cAMP and GDP are all ineffective. NADH shows a pattern of inhibition similar to that of ADP, though the apparent KI is higher by a factor of 200. NADPH relieves the inhibition by NADH. NAD+ also inhibits, b,t its affinity is a factor of 10 less than that of NADH. When ADP and NADH are added together, they act synergistically to inhibit the Ca2+-activated channel. It is concluded that the concept of the modification of enzyme activity by the allosteric binding of nucleotides, which is well established for soluble enzyme systems, also has application to the regulation of channels that control membrane permeability.
146 citations
TL;DR: The decrement in functional capacity of the nicotinic receptor on intact BC3H-1 cells has been simultaneously compared with the fractional occupation of the receptor by cobra alpha-toxin and a scheme for desensitization of the receptors is presented which incorporates both nonequivalence in the two agonist binding-sites and the maintenance of symmetry in the receptor states undergoing transitions.
130 citations
TL;DR: The results indicate that cells cultured from rat brain contain specific insulin receptors capable of mediating effects of insulin on macromolecular synthesis in the central nervous system.
111 citations
TL;DR: It is proposed that the cooperativity originates in ligand/ligand interactions in dimeric CRP, when bound to double-stranded deoxyribonucleic acid (dsDNA), CRP binds cAMP more efficiently, and the cooperateativity is positive even in conditions of low ionic strength where it is negative for the free protein.
Abstract: The binding of adenosine cyclic 3',5'-monophosphate (cAMP) and guanosine cyclic 3',5'-monophosphate (cGMP) to the adenosine cyclic 3',5'-monophosphate receptor protein (CRP) from Escherichia coli was investigated by equilibrium dialysis at pH 8.0 and 20 degrees C at different ionic strengths (0.05--0.60 M). Both cAMP and cGMP bind to CRP with a negative cooperativity that is progressively changed to positive as the ionic strength is increased. The binding data were analyzed with an interactive model for two identical sites and site/site interactions with the interaction free energy--RT ln alpha, and the intrinsic binding constant K and cooperativity parameter alpha were computed. Double-label experiments showed that cGMP is strictly competitive with cAMP, and its binding parameters K and alpha are not very different from that for cAMP. Since two binding sites exist for each of the cyclic nucleotides in dimeric CRP and no change in the quaternary structure of the protein is observed on binding the ligands, it is proposed that the cooperativity originates in ligand/ligand interactions. When bound to double-stranded deoxyribonucleic acid (dsDNA), CRP binds cAMP more efficiently, and the cooperativity is positive even in conditions of low ionic strength where it is negative for the free protein. By contrast, cGMP binding properties remained unperturbed in dsDNA-bound CRP. Neither the intrinsic binding constant K nor the cooperativity parameter alpha was found to be very sensitive to changes of pH between 6.0 and 8.0 at 0.2 M ionic strength and 20 degrees C. For these conditions, the intrinsic free energy and entropy of binding of cAMP are delta H degree = -1.7 kcal . mol-1 and delta S degree = 15.6 eu, respectively.
104 citations
TL;DR: The thermodynamics of receptor association with the salt-labile and salt-resistant nuclear-bound receptors appear similar and the evidence does not support the idea that these receptors have fundamental differences; the incomplete extraction of the receptors with salt may reflect some property of the solubilization process rather than two types of nuclear acceptors.
100 citations
TL;DR: Binding of [3H]diazepam is linear with platelet number, is temperature sensitive with maximum binding at 0°C, and has a broad optimal pH range between pH 5–9.
TL;DR: The non-linearity of the Scatchard type plots and slopes of the Hill plots of the binding data indicate that inducer binding exhibits cooperative behavior in the presence of operator DNA fragments, while free repressor binds inducer in a noncooperative fashion.
TL;DR: These studies demonstrated a widespread distribution throughout the animal kingdom of specific binding sites for kainic acid in neural tissue; the characteristics of these receptor sites are remarkably similar from primitive vertebrates to man.
TL;DR: Electron spin resonance studies of Triton X binding to BSA, covalently spin-labeled with N-(2,2,6,6-tetramethyl-piperidinyl-1-oxy)maleimide, indicated that the protein conformation in the vicinity of the labeled sulfhydryl was insensitive to NIS binding from dilute monomeric solutions.
Abstract: The binding of a series of Triton X nonionic surfactants (NIS) tobivine serum albumin (BSA) has been studied by equilibrium dialysis and titration calorimetry. At pH 7.0, Triton X molecules bind to two classes of sites, the first 2 molecules binding with positive cooperativity to high-affinity sites following by the binding of approximately 15 additional molecules to lower affinity, thermodynamically identical, and independent sites. The strength of the binding decreases as the number of oxyethylene units is increased in the surfactants Triton X-114, X-100, X-102, and X-165. Calorimetric measurements show the enthalpy change for the NIS-BSA interaction to be small and endothermic. Increasing the hydrophilic oxyethylene chain length results in a more endothermic enthalpy change and a smaller association constant. Electron spin resonance studies of Triton X binding to BSA, covalently spin-labeled with N-(2,2,6,6-tetramethyl-piperidinyl-1-oxy)maleimide, indicated that the protein conformation in the vicinity of the labeled sulfhydryl was insensitive to NIS binding from dilute monomeric solutions. Calorimetric experiments near the critical micelle concentration indicate, however, that the protein probably undergoes a conformational change associated with the population of the lower affinity NIS binding sites.
TL;DR: An improved purification procedure for the alpha 2 beta 2 complex of tryptophan synthase from Escherichia coli has been developed, and leads to material with a higher specific activity than reported previously.
Abstract: An improved purification procedure for the alpha 2 beta 2 complex of tryptophan synthase from Escherichia coli has been developed. It consists of DEAE-Sephacel chromatography, followed by hydrophobic chromatography on Sepharose CL 4B, and leads to material with a higher specific activity than reported previously. Inhibition studies, equilibrium dialysis, and spectrophotometric titration were used to study the binding both of pyridoxal phosphate analogues and of bisubstrate analogues. Pyridoxine 5'-phospate and N-phosphopyridoxyl-L-serine bind to the enzyme, but pyridoxamine 5'-phoshate and N-phosphopridoxyl-L-alanine do not. N-Phosphopyridoxyl-L-tryptophan is bound only weakly, although L-tyrptophan binds strongly to the alpha 2 holo beta 2 complex. It is likely that either differences is protonation or in geometry are responsible for the low affinity of the bisubstrate analogues in comparison to that of the external aldimines of either L-serine or L-tyrptophan with pyridoxal 5'-phosphate. As previously found with pyridoxal 5'-phosphate, pyridoxine 5'-phosphate, and N-phosphopryidoxyl-L-serine bind noncooperatively to two identical binding sites in the alpha 2 apo beta 2 complex. The same ligands bind with positive cooperatively to two binding sites in the apo beta 2 subunit. Because the analogues mimic the binding behavior of pyridoxal 5'-phosphate to both proteins, the internal aldimine of pyridoxal 5'-phosphate to the lysine amino group contributes only to the strength of that binding. The nickel apo beta 2 subunit, which is produced by limited proteolysis with trypsin, binds pyroxine 5'-phosphate noncooperatively to two identical sites. Therefore, the loop of polypeptide chain connecting the two autonomous domains of folding must be intact for enzyme activity, for the binding of the alpha subunit, and for cooperative binding of pyridoxine 5'-phosphate.
TL;DR: It is shown that GP32 can bind to nucleic acid lattices in two different conformations, and to characterize the molecular details of these binding species, and that these GP32-nucleic acid binding measurements can be used to provide a quantitative molecular interpretation of the sequential (competitive) binding equilibria involved in the autogenous translational regulation of GP32 synthesis.
TL;DR: Scatchard plots of binding of 125I-calmodulin to ghosts were consistent with the presence of a single class of high-affinity binding sites with the properties expected of (Ca2+ + Mg2+)-ATPase molecules and the binding showed positive cooperativity and occurred only in the absence of Ca2+.
TL;DR: Specific concentrations of concanavalin A (Con A), a lectin which mimics insulin activity, are shown here to inhibit one population of receptors leaving another population unaffected.
Abstract: Human erythrocytes specifically bind 125I-insulin in a manner similar to cells in which insulin exerts a physiological response1. In addition, erythrocytes are of practical value for correlating in vitro insulin binding with in vivo carbohydrate intolerance2,3. The competitive binding of labelled and unlabelled insulin to erythrocyte receptors is typically curvilinear when plotted according to Scatchard4. The curvilinear nature of the Scatchard plot describing insulin binding to membrane receptors, although originally attributed to heterogeneous sites5, has been more recently interpreted as negative cooperativity between homogeneous sites6. Evidence reported here, however, suggests that there are two populations of insulin receptors on erythrocytes. Specific concentrations of concanavalin A (Con A), a lectin which mimics insulin activity7, are shown here to inhibit one population of receptors leaving another population unaffected.
TL;DR: The evidence suggests that 125I-TSH binds to heterogeneous binding sites (of which the high affinity is probably the receptor for TSH) and that the enhanced dilution-induced dissociation of bound hormone by native hormone for this system, is only a characteristic of the low affinity binding site.
TL;DR: Using stopped-flow technique, a comparison of the rate constants, determined in the presence of an excess of cytochrome c, reveals the existence of two slower reacting sites on the functional unit of cy tochrome aa3, which is favoured in the non-cooperative model.
TL;DR: Rabbit anti‐SII pneumococcal polysaccharide IgG antibody was cross‐linked by dithiobis (succinimidyl propionate) and resembled two Y‐shaped structures connected in random orientations, but the data do not allow a clear distinction between noncooperative and cooperative binding.
Abstract: Rabbit anti-SII pneumococcal polysaccharide IgG antibody was cross-linked by dithiobis (succinimidyl propionate). The IgG dimers were visualized by electronmicroscopy and resembled two Y-shaped structures connected in random orientations. The three arms of the Y's were of equal length. The six globular domains of the IgG protomers could be seen, but the Fab arms and Fc stems could not be distinguished from each other. Electron microscopy revealed that the dimers were bound to the globular heads of C 1 q. From the dependence of thee weight average molecular weight on dimer concentration, and equilibrium constant of about 10(6) M(-1) was derived for the binding of dimers to C 1 q. The number of IgG dimers which could be accommodated at a single C 1 q molecule was derived to be three. The data do not allow a clear distinction between noncooperative and cooperative binding. The binding equilibrium was independent of whether the IgG dimers were liganded with an SII nonasaccharide hapten or not. The results are at variance with an allosteric mechanism of the action of antigen in C1 activation. They lend support to the association hypothesis of complement activation. The data suggest that clusters of about six IgG molecules, connected by a multivalent antigen or arranged at the cell surface, are recognized by C 1 q with a binding constant of about 10(10) M(-1).
TL;DR: The binding of the β subunit of Nerve Growth Factor to membrane preparations of 8-day chick embryo dorsal root ganglia (DRG) has been investigated under conditions similar to those used to study the binding of sNGF to intact single cell dissociates of DRG23, revealing heterogeneity of binding that is more complex than that seen with intact cells.
TL;DR: Owing to the highly cooperative binding, these differences are sufficient so that actin filaments nearly completely covered with uncross-linked tropomyosin are uncovered almost completely by cross-linking tropomyOSin at cysteine 190.
TL;DR: Direct binding studies show a parallel increase in the affinity of NADPH, confirming the previous notion that half-of-the-sites reactivity of the affinity label bromopyruvate is confirmed.
Abstract: Malic enzyme (ME) from pigeon liver is a tetrameric protein containing apparently identical subunits. In the present study, equilibrium dialysis and fluorescence titration techniques are employed to determine the binding parameters of nucleotide cofactors, malate, and the inhibitor oxalate. ME binds NADP+ or NADPH at four independent and equivalent sites with dissociation constants of 1.33 microM (pH 7.5, 4 degrees C) and 0.29 microM (pH 7.0, 5 degrees C), respectively, showing "all-of-the-sites" reactivity. The affinity of both nucleotides decreases with increasing temperature, yielding delta Hdissociation values of 11.4 kcal/mol for E-NADP+ and 8.9 kcal/mol for E-NADPH, thus implicating the involvement of polar forces in the binding process. The affinity of NADP+ is independent of pH between 6.1 and 8.4 whereas that of NADPH is highly pH dependent and decreases approximately 63-fold from pH 6.0 to pH 8.0. The pH profile suggests the participation of a protonated enzyme group(s) (pK = 7.2-7.5) in NADPH binding, probably a histidine residue. The affinity of NADP+ is enhanced ca. twofold by pyruvate, in the presence of Mn2+ (50-100 microM) saturating only two "tight" metal sites [Hsu, R. Y., Mildvan, A. S., Chang, G. G., & Fung, C. H. (1976) J. Biol. Chem. 251, 6574]. Binding of Mn2+ at weak metal sites (KD congruent to 0.9 mM) prevents this change. Malate binds free ME or binary E-Mn2+ and E-NADP+ (H) complexes weakly with dissociation constants of greater than or equal to 2 mM. The affinity is significantly increased by Mn2+ and NADPH in the ternary E-Mn2+-NADPH complex, yielding two "tight" (KD = 22-30 microM) and two "weak" (KD = 250-400 microM) malate sites per enzyme tetramer as the result of either preexisting nonidentity or negative cooperativity between intitially identical sites. The transition-state inhibitor oxalate binds ME tightly (KD = 65 microM) at the two tight malate sites, showing "half-of-the-sites" stoichiometry. The binding parameters are unaffected by Mn2+, whereas the affinity of this inhibitor is enhanced 3.5-fold by saturation with NADPH. Further evidence for the half-of-the-sites reactivity of the affinity label bromopyruvate [Pry, T. A., & Hsu, R. Y. (1978) Biochemistry 17, 4024] is obtained by sequential modification of the four putatively identical SH groups of ME with bromopyruvate, 5,5'-dithiobis(2-nitro-benzoic acid), and K14CN. The modified enzyme has a structure of E4(S-pyr)2(S-14CN)2 and is "inactive" in the reaction with malate. In contrast, the E(S-14CN)4 derivative prepared in the absence of bromopyruvate is completely active. The oxidative decarboxylase reaction is inhibited by high concentrations (greater than or equal to 0.3 mM) of malate in the presence of tightly bound Mn2+. Direct binding studies show a parallel increase in the affinity of NADPH, confirming our previous notion [Reynolds, C. H., Hsu, R. Y., Matthews, B., Pry, T. A., & Daibits the rate-limiting NADPH release step.
TL;DR: The data suggest that whole brain contains at least three major muscarinic receptors, which can be distinguished on the basis of their affinities for agonists.
TL;DR: A Ca2+-dependent protein phosphatase inhibition like that which was shown earlier in the protein-glycogen complex can be observed in the sarcoplasmic reticulum andKinetically a competitive type of inhibition can be characterized and this inhibition can only be observed at high protein concentrations.
TL;DR: Analysis of the data was consistent either with receptors with different affinities or with a single class of receptors, which demonstrates negative cooperativity, and the binding and degradation of insulin were dependent on temperature and duration of incubation as well as on the tubule concentration.
Abstract: Binding and degradation of monoiodinated [125I]insulin by isolated tubules of rat kidney cortex were studied. Binding and degradation of insulin were dependent on temperature and duration of incubation as well as on the tubule concentration. Degradation was minimized by the use of low to moderate tubule concentrations and an incubation temperature of 22 C. Increasing concentrations of unlabeled insulin (0.1–1000 nm added in the incubation medium) decreased [125I]insulin binding in a dose-dependent fashion. Analysis of the data was consistent either with receptors with different affinities or with a single class of receptors, which demonstrates negative cooperativity. Parathyroid hormone and other peptide hormones did not affect [125I]insulin binding to tubules. Approximately 70–75% of bound [125I]insulin could be dissociated by the subsequent addition of excess unlabeled insulin. Insulin binding preceded degradation, and preincubation of tubules with trypsin abolished binding and degradation of insulin. T...
TL;DR: Rifampicin is shown to have a slight stimulatory effect on the extent of open complex formation of the core enzyme, and the presence of the sigma subunit significantly altered the nature and extent ofopen complex formation.
TL;DR: The results are consistent with the view that the glycoprotein is the site of regulation of Ca2+ equilibration across the mitochondrial membrane and make it possible to conclude that the effector in the phenomenon is NAD+.
Abstract: A change in the redox state of pyridine nucleotides such as that evoked by addition of oxalo-acetate has been shown to promote Ca2+ efflux from Ca2+ pre-loaded respiring mitochondria. An affinity-chromatography-purified antibody preparation obtained against the mitochondrial Ca2+-binding glycoprotein inhibits the phenomenon. This finding suggests that the glycoprotein is involved also in the oxaloacetate-induced Ca2+ release. This conclusion is reinforced by the finding that Ca2+-binding glycoprotein shows four sites per molecule where the pyridine nucleotides may be bound. Binding of NAD+ occurs preferentially over the others and the binding shows positive cooperativity, indicating that the glycoprotein undergoes an allosteric change upon NAD+ binding. Interestingly, in addition, NAD+ lowers the affinity of the glycoprotein for Ca2+. The effect cannot be induced by NADH. Pyridine nucleotide phosphates, NADP+ and NADPH, are essentially not bound. The results are consistent with the view that the glycoprotein is the site of regulation of Ca2+ equilibration across the mitochondrial membrane and make it possible to conclude that the effector in the phenomenon is NAD+.
TL;DR: The binding of 2-nonanone to bovine serum albumin exhibited positive cooperativity at low molal ratios of binding, indicating stabilization of the hydrophobic binding sites at low concentrations of 2 nonanone, and the degree of cooperativity was affected by the type of anion present.