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Showing papers on "Cooperative binding published in 1987"


Patent
02 Sep 1987
TL;DR: In this article, a single polypeptide chain binding molecule has been proposed which has binding specificity and affinity substantially similar to the binding specificity of the light and heavy chain aggregate variable region of an antibody.
Abstract: The invention pertains to a single polypeptide chain binding molecule which has binding specificity and affinity substantially similar to the binding specificity and affinity of the light and heavy chain aggregate variable region of an antibody, to genetic sequences coding therefor, and to recombinant DNA methods of producing such molecule and uses for such molecule.

3,290 citations


Journal ArticleDOI
TL;DR: The experimental results are inconsistent with the one- site model but are explained by a two-site model in which the ternary complexes of So .

105 citations


Journal ArticleDOI
TL;DR: Assembly of the extrinsic pathway on cell surfaces was investigated by studying the binding and activity of factor VII on the bladder carcinoma cell line J82 which expressed 18,800 milliunits of tissue factor activity/10(6) cells.

73 citations


Journal ArticleDOI
TL;DR: The results indicate that the aminoterminal dimerization site of cGMP-dependent protein kinase and the autophosphorylation site, present in this part, control not only the activation of the enzyme but also the cooperative binding characteristics of the intact enzyme.
Abstract: Treatment of cGMP-dependent protein kinase with low concentrations of trypsin generates an enzyme fragment of 65 kDa which is fully active in the absence of cGMP. The fragment has a s20,w value of 4.6 S indicating that the active fragment is a monomer of 65 kDa. Trypsin removes the first 77 amino acids which contain the aminoterminal dimerization site and the autophosphorylation sites. The Km and Vmax values of the fragment for ATP and Kemptide were essentially the same as those for the native enzyme. The fragment binds 2 mol cGMP/mol fragment with affinities close to that of the native enzyme. However, binding of cGMP to these sites was non-cooperative and shows similar characteristics to the autophosphorylated native enzyme. These results indicate that the aminoterminal dimerization site of cGMP-dependent protein kinase and the autophosphorylation site, present in this part, control not only the activation of the enzyme but also the cooperative binding characteristics of the intact enzyme.

62 citations


Journal ArticleDOI
TL;DR: A model in which troponin/tropomyosin (Tn/Tm) controls the actin-S1 interaction by inhibiting the isomerization step is proposed and can account for the cooperative binding of S1 and S1 nucleotide complexes to actin.

46 citations


Journal ArticleDOI
TL;DR: The binding characteristics of the GlcNAc binding protein present in thyroid membranes were reinvestigated using neoglycoproteins as probes and it is suggested that this Glc NAc receptor functions in thyroglobulin metabolism, possibly involved in recycling of internalized thyrogLobulin molecules back into the follicular lumen.

41 citations


Journal ArticleDOI
TL;DR: Data indicate that the fluorescence change is a direct measure of the S1-induced change of state of Tm in the fully reconstituted thin filament.(ABSTRACT TRUNCATED AT 250 WORDS)
Abstract: The monomer fluorescence of N-(1-pyrenyl)maleimide-labeled tropomyosin bound to F-actin (PTm-actin) increases when myosin subfragment 1 (S1) binds to actin and is half complete when only approximately 1 S1 is bound to 7 actin subunits [Ishii, Y., & Lehrer, S. S. (1985) Biochemistry 24, 6631-6638]. Similar studies of the binding of S1 and S1-ADP to fully reconstituted thin filaments [PTm-actin-troponin (Tn)] are now reported. The pyrene monomer fluorescence change was half complete when approximately 0.5 S1/7 actin subunits and approximately 1.5 S1/7 actin subunits were bound in the presence and absence of Ca2+, respectively. In the presence of Mg2+-ADP, when S1 binding is weakened, the S1 binding profiles and fluorescence changes were sigmoidal, with the cooperative transitions occurring at lower [S1] in the presence of Ca2+ as first shown by Greene and Eisenberg for S1 binding [Greene, L., & Eisenberg, E. (1980) Proc. Natl. Acad. Sci. U.S.A. 77, 2616-2620]. It was possible to fit both the binding and fluorescence data with the same parameters of a two-state (weak and strong S1 binding) cooperative binding model [Hill, T., Eisenberg, E., & Greene, L. (1980) Proc. Natl. Acad. Sci. U.S.A. 77, 3186-3190] for each Ca2+ situation if the fluorescence change is interpreted as the fraction of tropomyosin (Tm) units in the strong S1 binding state. These data indicate that the fluorescence change is a direct measure of the S1-induced change of state of Tm in the fully reconstituted thin filament.(ABSTRACT TRUNCATED AT 250 WORDS)

39 citations


Journal ArticleDOI
TL;DR: Evidence is presented that the pairwise arrangement of characteristic helix-loop-helix calcium-binding sites can result in the positive cooperative binding of Ca2+.
Abstract: The large calcium gradient across the plasma membrane creates different environments for intra- and extracellular calcium-binding proteins. The latter are continuously surrounded by 10(-3) M Ca2+, which promotes activation or stabilization of certain proteases, nucleases, or lipases. Other proteins, such as those involved in blood clotting, contain polyelectrolyte regions that are composed of carboxyglutamic or phosphoserine moieties that allow them to interact with Ca2+. In contrast, intracellular calcium-binding proteins, such as calmodulin and troponin C, the trigger proteins for muscle contractions, need to respond to an increase in Ca2+ from 10(-7) to 10(-6) M during cell activation. Evidence is presented that the pairwise arrangement of characteristic helix-loop-helix calcium-binding sites can result in the positive cooperative binding of Ca2+. This can be further promoted by the binding of ligands, drugs, or target proteins Several drug binding sites on calmodulin are allosterically related and their localization on the unusual dumbbell structure of this molecule will be discussed.

30 citations


Journal ArticleDOI
TL;DR: It is suggested that the low‐affinity binding state of the [3H]HC‐3 binding sites represents the „functional” form for the SDHACU system, and that unlabeled HC‐3 selectively labels SDH ACU sites located on presynaptic cholinergic neurons in rat CNS.
Abstract: The characteristics of [3H]hemicholinium-3 ([3H]HC-3) interactions with rat striatal membranes were investigated. Under the described assay conditions, [3H]-HC-3 binds with a saturable population of membrane binding sites having the following regional distribution: striatum much greater than hippocampus greater than or equal to cerebral cortex greater than cerebellum. The specific binding of [3H]HC-3 showed an obligatory requirement for NaCl; other halide salts of sodium or KCl failed to substitute for NaCl. The Scatchard transformation of saturation isotherm data generated a curvilinear plot with high- and low-affinity components of binding. The dissociation of [3H]HC-3 at infinite dilution was also multiexponential. The dissociation could, however, be accelerated if unlabeled HC-3 was included in the diluting buffer, and this increase in dissociation appeared to be dependent on the concentrations of unlabeled HC-3 used, with the maximal increase demonstrable at 100 nM. The dissociation was also dependent on the fractional saturation of binding sites with labeled HC-3, such that, at higher fractional saturation of binding sites, the overall dissociation was faster and the difference in the dissociation observed between "dilution only" and "dilution + unlabeled HC-3" was reduced. This occupancy-dependent change in dissociation could also be influenced by temperature and pH. Based on the results of these kinetic studies, the steady-state [3H]HC-3 binding data were analyzed for a homogeneous population of binding sites undergoing site-site interactions of the negative cooperative type. Such an analysis yielded a KD of 9.3 nM for the high-affinity state and a KD of 22.8 nM for the low-affinity state of binding sites, with a Bmax of 434 fmol/mg of protein. Competitive binding studies showed that unlabeled HC-3 was most potent in displacing [3H]HC-3, followed by choline. Other drugs known to have little influence on the synaptosomal sodium-dependent high-affinity choline uptake system (SDHACU) had no significant effect on [3H]HC-3 binding sites. Similarities in ionic dependencies, regional distributions, and pharmacological selectivities of [3H]HC-3 binding with synaptosomal SDHACU suggest that [3H]HC-3 selectively labels SDHACU sites located on presynaptic cholinergic neurons in rat CNS. We suggest that the two affinity states of [3H]HC-3 binding sites represent the different "functional" states of the SDHACU system. The binding of HC-3 (or choline) with the high-affinity state of the binding sites induces negative cooperative site-site interactions among the binding sites, resulting in the formation of a low-affinity binding state.(ABSTRACT TRUNCATED AT 400 WORDS)

28 citations


Journal ArticleDOI
TL;DR: It is concluded that the inhibitor binding sites are involved in proton translocation in F1-ATPase, which allosterically induce the correct structure of the rhodamine 6G binding site.

26 citations


Journal ArticleDOI
TL;DR: Analysis of the enzymatic activation of myosin light chain kinase at different concentrations of calmodulin and Ca2+ revealed that this Ca2-free complex is inactive and that activation is concomitant with the formation of the enzyme.

Journal ArticleDOI
TL;DR: The binding of3H-acetylcholine to nicotinic receptors in rodent and human brain was measured in the presence of atropine to prevent binding to muscarinic binding sites, finding that there appears to exist multiple binding sites for 3H-ACh in human cerebral cortex.
Abstract: The binding of3H-acetylcholine (3H-ACh) to nicotinic receptors in rodent and human brain was measured in the presence of atropine to prevent binding to muscarinic binding sites.3H-ACh binds specifically and saturably to rodent brain. From saturation binding Kd was 30 nM in rat cerebral cortex, which is close to that calculated from kinetic experiments. The binding was temperature-dependent, being highest at low temperatures and decreasing at higher temperatures. The regional distribution of binding in mouse brain was not uniform. The binding was highest in the midbrain, intermediate in the cerebral cortex and striatum, and lowest in the cerebellum, hippocampus, hypothalamus and medulla oblongata. No significant correlation was found between the regional3H-ACh binding and the regional binding of3H-alpha-bungarotoxin (3H-BTX),3H-nicotine (3H-NIC),3H-tubocurarine and the endogenous acetylcholine content, although the correlation value for3H-ACh/3H-NIC was at the limit for significance.3H-ACh also bound specifically to human cerebral cortical tissue and this binding was approximately three times lower than in rodent brain, when a low3H-ACh concentration was used. In contrast to rat brain there appears to exist multiple binding sites for3H-ACh in human cerebral cortex as suggested by the curvelinear nature of the Scatchard plot. It was calculated that3H-ACh bound with Kd 4 nM and Bmax 8 pmol/g protein and Kd 112 nM and Bmax 67 pmol/g protein. The Hill number of 1.5 for the binding of low concentration and 2.5 for high concentration of3H-ACh also suggest that the3H-ACh-binding sites interaction exhibit positive cooperativity.

Journal ArticleDOI
TL;DR: Fluorometric titration of E. coli single-stranded DNA binding protein with various RNAs showed that the protein specifically and cooperatively binds to its own mRNA.
Abstract: Fluorometric titration of E. coli single-stranded DNA binding protein with various RNAs showed that the protein specifically and cooperatively binds to its own mRNA. The binding inhibited in vitro expression of ssb and bla but not nusA. This inhibition takes place at a physiological concentration of SSB. The function of the protein in gene regulation is discussed.


Journal ArticleDOI
TL;DR: The data indicate that most of the interactions between C1q and C1R2C1s2 originates from a strong binding to the C1r2 moiety of the zymogen complex, which is lost upon activation of C1 r2.


Journal ArticleDOI
TL;DR: Investigation of microvilli isolated from 13762 mammary ascites tumor cells finds a major calcium‐sensitive protein (AMV‐p35) that can be isolated with microvillar microfilament cores prepared by Triton X‐100 extraction in the presence but not absence of calcium.
Abstract: Microvilli isolated from 13762 mammary ascites tumor cells contain a major calcium-sensitive protein (AMV-p35) that can be isolated with microvillar microfilament cores prepared by Triton X-100 extraction in the presence but not absence of calcium. AMV-p35 can be readily purified from ethylene glycol bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid extracts of the microfilament cores by chromatography on an anion exchange column, to which it does not bind. Immunoblot analysis indicates that AMV-p35 is related to calpactin I, the pp60src tyrosine kinase substrate. In the presence of calcium, AMV-p35 binds approximately 4 mol of chlorpromazine per mole of protein in a binding process showing apparent positive cooperativity, similar to calmodulin; however, in contrast to calmodulin, AMV-p35 also binds phenothiazine in the absence of calcium.

Book ChapterDOI
TL;DR: In this article, the formation of oligonuclear complexes designated as Cd4- and Cd3-cluster has been monitored in MT reconstituted with varying amounts of Cd using differential modification of Cys with 14Ciodoacetamide.
Abstract: Mammalian metallothioneins (MT) contain 20 Cys in a total of 61 amino acid residues and bind 7 Cd and/or Zn ions. The metal is localized in two clusters made up of three and four metal-thiolate complexes in the NH2- and COOH-terminal half of the chain, respectively (1). The formation of these oligonuclear complexes designated as Cd4- and Cd3-cluster has now been monitored in MT reconstituted with varying amounts of Cd using differential modification of Cys with 14C-iodoacetamide. At ratios below 3 moles Cd/mole MT bound, no differential protection of Cys by the metal and, hence, no preferred binding is detectable. At Cd-to-protein ratios between 3 and 5 moles Cd/mole MT the modification profiles reveal preferred and cooperative binding in the COOH-terminal half of the chain indicating formation of the Cd4- cluster. At still higher ratios formation of the Cd3-cluster is initiated in the NH2-terminal section of the polypeptide chain. Comparison of the differential modification data of Cd6-MT and Cd7-MT suggests that the last Cd to be bound is coordinated to Cys ligands located mainly between positions 20 and 30 of the sequence. The extent of labelling of the different Cys in Cd7-MT indicates that the ligands of the Cd3-cluster are three times as accessible to iodoacetamide as those of the Cd4-cluster, suggesting a greater thermodynamic stability of the latter.

Book ChapterDOI
01 Jan 1987
TL;DR: These studies suggest that both (-)-and (+)-nicotine are involved in complex binding site and receptor interactions which may involve positive cooperativity as well as up-regulation of binding at the high affinity sites.
Abstract: These studies suggest that both (-)-and (+)-nicotine are involved in complex binding site and receptor interactions which may involve positive cooperativity as well as up-regulation of binding at the high affinity sites. Early studies employing (±)-[3H]nicotine as the labeled ligand suggested that (-)-and (+)-nicotine each bound to 3 rat brain membrane sites: a very high affinity site, a high-affinity site and a low-affinity site. It was also found that each of the isomers enhanced the binding of (±)-[3H]nicotine in low concentrations and that (+)-nicotine was more potent and efficacious in producing this effect than (-)-nicotine. Further, both the configuration of Scatchard plots of saturation curves as well as Hill plots at low concentrations (nH = 1.34) suggested positive cooperativity. In subsequent studies employing optically pure (-)-and (+)-[3H]nicotine it was found in saturation studies that (+)-nicotine occupied only one-tenth as many binding sites as (-)-nicotine. This suggested that (+)-nicotine might be occupying a site different from most of the sites occupied by (-)-nicotine. It was further observed that (-)-and (+)-nicotine increased the binding of both (-)-and (+)-[3H]nicotine. (+)-Nicotine was more effective in this regard than (-)-nicotine. In an effort to identify drugs with specificity for nicotine’s binding sites, the binding characteristics of over 50 substituted pyridines, piperidines, and pyrrolidines, as well as other nicotinic drugs, were studied using (±)-[3H]nicotine as the labeled ligand. Drugs were found to differ markedly both in their ability to inhibit as well as to enhance the binding of (±)-[3H]nicotine. Compounds were identified which had specificity for the up-regulatory site in that they enhanced binding over a wide range of concentrations but had little ability to inhibit binding. Of these (±)-2-methylpiperidine was the most specific. Its activity in enhancing the binding of (-)-[3H]nicotine resides in its (+)-isomer whereas both isomers are equally effective in inhibiting the binding of (-)-and (±)-[3H]nicotine. Saturation studies with (±)-[3H]-2-methylpiperidine indicate that it binds to a very high affinity binding site. These data taken together suggest that the very high affinity nicotine binding site is probably the up-regulatory site (Site I) and have identified 2 drugs with stereospecificity for this site, (+)-2-methylpiperidine and (+)-nicotine. The data further suggest that there are separate (-)-[Site II(-)] and (+)-nicotine [Site III(+)] high-affinity binding sites and a low-affinity site (Site IV).

Journal ArticleDOI
TL;DR: From the data, it is suggested that paraquat binds primarily to the negatively charged phosphates on the DNA backbone but is displaced into the interbase region occupied by the intercalator ethidium bromide.

Journal ArticleDOI
TL;DR: The present paper deals with one such enzyme, the DHFR from C. albicans, where it is shown that the binding of substrates exhibit strong negative cooperativity, and determines the relationship between inhibitor/NADPH cooperativity and the relative insensitivity of N. gonorrhoeae to TMP.

Journal ArticleDOI
TL;DR: The nonspecific interaction of the mitogenic lectin Concanavalin A with glycosyl‐free liposomes of various composition has been investigated by microcalorimetric titration measurements and appears to represent the main driving force for the strong attachment of the lectin to membrane surfaces.
Abstract: The nonspecific interaction of the mitogenic lectin Concanavalin A (Con A) with glycosyl-free liposomes of various composition has been investigated by microcalorimetric titration measurements. The results obtained show the following features of main interest: (1) the affinity constants (Ka) of the interaction of Con A with liposomal bilayers are in the order of magnitude 105–106M−1. The reaction enthalpies (ΔH) are positive, and small (approximately 0.1 KJ mol−1 lipid), compared to the free energy terms (−ΔG = 30–40 KJ mol−1 lipid). All lectin–lipid interactions are strongly entropy-controlled (ΔH/TΔS < 1.0). These thermodynamic features are characteristic for hydrophobic interaction processes. (2) The liposomal head-group charge does not significantly affect the lipid-affinity of Con A. Electrostatic forces thus appear to play a minor role in lectin–lipid interactions. (3) The lipid affinity of Con A is sensitive to the fluidity of the liposomal bilayers, increasing with increasing fluidity. Below the gel to liquid-crystal phase transition temperature, the lectin binding to liposomal bilayers is inhibited. (4) The binding isotherms, corresponding to the interaction of Con A with liposomes, composed of tightly packed, saturated phospholipids, exhibit pronounced positive cooperativity. This phenomenon is absent in the binding curves, corresponding to the interaction of Con A with more fluid liposomal bilayers. (5) The Con A specific inhibitor α-D-methylmannopyranoside (50 mM) drastically increases the molar reaction enthalpy. The Ka term is significantly reduced in presence of the inhibitor sugar. Urea induces analogous changes in the thermodynamic parameters of the lectin–lipid interaction. The effects of α-D-methylmannopyranoside are thus not Con A specific, but are attributable to solvent effects. (6) It was shown that the binding of one Con A molecule affects a large number (approximately 1000) of phospholipid molecules in the liposomal bilayer. (7) The affinity constants (Ka) of the interaction of Con A with glycosyl-free lipids are smaller by a factor of approximately 10, compared to the Ka terms, reported for Con A binding to biological membranes. The presence of glycosidic receptor groups thus controls the specificity of lectin–membrane interactions, whereas the nonspecific lectin–lipid interactions appear to represent the main driving force for the strong attachment of the lectin to membrane surfaces.

Journal ArticleDOI
01 Jan 1987
TL;DR: The theory derived for case (a) is applied to the binding of fluoresceinated epidermal growth factor to A431 cells and it is demonstrated that the analysis of data obtained from both conventional and competition assays provides information which is difficult, if not impossible, to obtain from either assay alone.
Abstract: A common assumption invoked in the analysis of competition binding assays is that the fractional saturation of sites with the unlabeled ligand is given by 1 - (the concentration of bound labeled ligand in the presence of unlabeled ligand)/(the concentration of bound labeled ligand in the absence of unlabeled ligand). This assumption is critically evaluated in the context of several binding models: (a) binding of univalent ligands to multiple classes of equivalent and independent sites, with and without nonspecific binding; (b) cooperative binding of univalent ligands; and (c) binding of multivalent ligands to a single class of univalent acceptors. We show that the conventional assumption is only valid when the labeled ligand is mainly in the free form, occupies a small fraction of the total sites and binds univalently to all sites in an equivalent and independent manner, and when the unlabeled ligand forms l : l complexes with the acceptor sites. When these conditions are satisfied, the convention...

Journal ArticleDOI
TL;DR: The binding of high-density lipoproteins (HDL3) to tissue culture dishes is studied as an example of binding without biological significance, indicating that computer-assisted analysis, while most accurately describing binding data, nevertheless does not ensure that measured specific binding has biological significance.

Journal ArticleDOI
TL;DR: It is concluded that the idiotope is restricted to the monomer 2 type of the Mcg lambda chain conformational isomer, and probably involves more than one linear sequence since reduction and alkylation of the intra- and inter-chain disulphide bonds in 8 M urea leads to a complete loss of binding of the anti-idiotype.

Journal ArticleDOI
TL;DR: It is shown, using DNase footprinting experiments with purified A protein, that mutant A binding sites, which affect transposition, have decreased affinity for the transposase.
Abstract: Transposition of the E. coli bacteriophage Mu requires the phage encoded A and B proteins, the host protein HU and the host replication proteins. The ends of the genome of the phage, on which some of these proteins act, both contain three transposase (A) binding sites. The organization of these binding sites on each end, however, is different. Here we show, using DNase footprinting experiments with purified A protein, that mutant A binding sites, which affect transposition, have decreased affinity for the transposase. Furthermore the transposase binds non-cooperatively to all A binding sites both in the left and right end of Mu. Electron microscopic studies show that the A protein forms specific nucleoprotein structures upon binding to the ends of Mu. The A and B proteins interact with the ends of Mu to generate larger structures than with the A protein alone.

Journal ArticleDOI
TL;DR: The so called “self regulation effect” between the isolated binding constant (intrinsic binding constant) and the cooperative binding constant, (binding constant of growth process) can be observed amongst the large amount of data so far reported.
Abstract: As for the cooperative binding of small molecules to the linear polymers, including not only synthetic polymers but also biopolymers, an interesting relationship, the so called “self regulation effect” between the isolated binding constant (intrinsic binding constant) and the cooperative binding constant, (binding constant of growth process) can be observed amongst the large amount of data so far reported. The biological significances as well as the possible origin for this “self regulation mechanism” will be discussed.

Book ChapterDOI
01 Jan 1987
TL;DR: CG-PK, a homo-dimer of 150 kDa, has four partially cooperative binding sites for cGMP with KD-values in the order of 10 to 200 nM as has been shown by binding studies with 3H-cGMP1,2.
Abstract: Cyclic GMP (cGMP) is a second messenger for cellular regulation and activates cGMP-dependent protein kinase (cG-PK). cG-PK, a homo-dimer of 150 kDa, has four partially cooperative binding sites for cGMP with KD-values in the order of 10 to 200 nM as has been shown by binding studies with 3H-cGMP1,2. Two types of sites have been described, site 1 with high affinity and slow dissociation and site 2 with lower affinity and faster dissociation. The primary structure of the enzyme has been reported and assigned to functional domains3.

Book ChapterDOI
01 Jan 1987
TL;DR: This work has compared structural properties, ligand binding and ATP hydrolysis of the holoenzyme with those of the isolated subunits of this enzyme complex with the changes that occur upon reconstitution of the bigger complexes.
Abstract: For a better understanding of the molecular mechanism of the H+-ATP-synthase, a prerequisite is to know more about the correlation between structural and functional aspects. One possibility to study this correlation is to compare the properties of the subunits of this enzyme complex with the changes that occur upon reconstitution of the bigger complexes. Because of its extreme stability, the ATP-synthase of the thermophilic bacterium PS3 is especially well suited for these kinds of experiments (1, 2). Using this enzyme we have compared structural properties, ligand binding and ATP hydrolysis of the holoenzyme with those of the isolated subunits. For experiments concerning ligand binding and functional properties, the adenine-nucleotide analogue naphthoyl-ATP (N-ATP) has been used (3, 4); the advantages of this ligand are: (1) being fluorescent, (2) having a high affinity for the ATP-binding sites, (3) affecting the intrinsic fluorescence of TF1, (4) being a potent inhibitor of the ATPase reaction. Preparation procedures and the methods have been described in detail elsewhere (5–7).

Book ChapterDOI
TL;DR: It is concluded from these results that Ca2-saturated C-domain bound to target enzyme induces a positive cooperative Ca2+ binding to N-domain that works as a regulatory domain.
Abstract: Publisher Summary This chapter presents the results of a study that analyzes regulatory and target-binding domains of calmodulin. Calmodulin can bind 4 mol Ca2+ per mol extending over 2 pCa units. To attain the maximum activity of calmodulin-dependent enzymes within a narrow concentration of Ca2+, it is expected for calmodulin to bind all 4 mol of Ca with positive cooperativity. In this study, the Ca2+ binding to calmodulin was measured in the presence of target proteins, mastoparan and caldesmon. Mastoparan increased Ca2+ affinity about 17-fold and the Ca2+ titration curve suggests an increase in the cooperativity. Caldesmon also increased the positive cooperativity, but the increase in the Ca2+ affinity was rather small (2.9-fold). The results of Ca2+ binding were compared with 1H nuclear magnetic resonance spectrum of calmodulin in the presence of equimolar concentration of mastoparan. It is concluded from these results that Ca2+-saturated C-domain bound to target enzyme induces a positive cooperative Ca2+ binding to N-domain that works as a regulatory domain.