The binding of calcium to a salivary phosphoprotein, protein A, common to human parotid and submandibular secretions.

The binding of Ca2+ to a previously described phosphoprotein from human parotid saliva, protein A [Bennick (1975) Biochem J. 145, 557-567] was studied by means of equilibrium dialysis. In 5 mM-Tris/HC1 buffer, pH7.5, protein A bound 664nmol of Ca/mg of protein. Km was determined to be 181 muM and the binding of Ca2+ to the protein was non-co-operative. The binding of Ca2+ apparently occurs to side-chain carboxyl groups in the protein, but protein phosphate is of minor if any importance in calcium binding. Hydrolysis of protein A by trypsin and collagenase or heating of the protein at 60 degrees or 100 degrees C did not affect Ca2+ binding. The Ca2+ binding decreases with increased concentration of the dialysis buffer and on the addition of SrCl2, or MgCl2 or MnCl2 to the dialysis buffer. Protein A does not aggregate in the presence of Ca2+, since the s20,w was identical when determined in the presence (1.30S) and absence (1.35S) of CaCl2. By use of a specific antiserum to protein A it was found that protein C [Bennick & Connell (1971) Biochem. J. 123, 455-464] and perhaps minor related components cross-reacted with protein A. No other salivary proteins showed immunological similarity. Proteins A and C were also present in submandibular saliva. The possible functions of protein A are discussed.     

Properties of calcium binding by Myxicola axoplasmic protein

The 45Ca2+ binding properties of axoplasmic protein from the Myxicola giant axon have been investigated using a centrifugal/concentration-dialysis technique. Scatchard plot analysis of these binding data suggest that Ca2+ is attached to a site with an equilibrium dissociation constant of 7.7 +/- 0.5 microM and a capacity of 4.4 +/- 0.2 mumol/g axoplasmic protein (n = 11). Addition of other cations--Cd2+, Mn2+, Al3+, Cu2+, Ba2+, and Zn2(+)--at concentrations up to 10 microM did not displace 0.2 microM 45Ca2+ from its binding site, probably because of buffering of these cations by amino acid residues within the protein solutions. The protein could be stored at 4 degrees C for up to 16 days with no appreciable change in the number of calcium sites. Ca2+ binding equilibrium took place in less than 30 min of incubation. Increasing the incubation temperature from 4 degrees C to 37 degree C reduced the number of Ca2+ sites. The binding capacity was reduced by one-half when the protein was dialyzed with 4 M urea or high ionic strength KCl (2 M). Calcium binding was examined as a function of pH. When the protein was dialyzed overnight at different pH values and all the binding was done at pH 7.0, the apparent number of Ca2+ sites decreased as the pH of the dialysis medium was increased. When the protein was dialyzed overnight at pH 7.0 and the binding was done at different pH values, the apparent binding capacity increased as pH increased.(ABSTRACT TRUNCATED AT 250 WORDS)     

Binding strength of calcium ion to bovine alpha-lactalbumin

A Ca2+-sensitive electrode was used for determination of the binding strength of Ca2+ to bovine alpha-lactalbumin in 60 mM Tris buffer (pH 7.8-8.5) in the presence of various concentrations of NaCl. The dependence of the apparent binding constant on the concentration of NaCl was consistent with competitive binding of Ca2+ and Na+, and the binding constants of Ca2+ and Na+ were found to be 2.2 (+/- 0.5) X 10(7) M-1 and 99 (+/- 33) M-1, respectively, at 37 degrees C and pH 8.0. The temperature dependence of the binding constant of Ca2+ was examined between 30 and 45 degrees C; extrapolation of the dependence led to a binding constant of approximately 1 X 10(8) M-1 at pH 8.4 and 25 degrees C. The electrostatic contribution and conformational effect of the protein were also taken into consideration, and the intrinsic binding constant of Ca2+ to native alpha-lactalbumin was calculated to be (1.2-1.5) X 10(10) M-1 at 37 degrees C and pH 8.0.     

Stoichiometry of manganese and calcium ion binding to concanavalin A

Using measurements of solvent nuclear (proton) magnetic relaxation dispersion (NMRD), we have previously shown that concanavalin A (Con A) can exist in two conformational forms and that, in the absence of Ca2+, Mn2+ can bind to both the S1 and S2 sites of each monomer of Con A of at least one conformer [Brown, R.D., III, Brewer, C.F., & Koenig, S.H. (1977) Biochemistry 16, 3883-3896]. Recently other investigators have claimed that the stoichiometry of Mn2+ binding to Con A is only 1:1 for this conformational state, both in the absence and presence of saccharide; the same was claimed for Ca2+ under similar conditions. We now present titration and equilibrium dialysis experiments, both in the absence and presence of saccharide, using NMRD and atomic absorption spectroscopy, to investigate the stoichiometry of Mn2+ and Ca2+ binding to Con A. We have extended the NMRD method to include the determination of the total concentration of Mn2+ in samples of Con A. This, coupled with our previous use of NMRD to measure the concentration of free Mn2+ in protein solutions as well as the distribution of bound Mn2+ among different sites, allows us to measure the stoichiometry of binding with precision. We reconfirm that, at equilibrium in the presence of excess Mn2+, the binding stoichiometry of Mn2+ to Con A is 2:1, both in the absence and presence of saccharide. Addition of Ca2+ to a solution of Mn2+-Con A results in stoichiometric displacement of Mn2+ from the S2 site under the conditions investigated. Under nonequilibrium conditions, Mn2+ forms a metastable binary complex with the protein that persists for days at 5 degrees C. We also report, for the first time, values for all of the dissociation constants of binary and ternary complexes of Mn2+ with both conformations of Con A in solution. Atomic absorption measurements also indicate that Ca2+, in the absence of Mn2+, binds to both S1 and S2 sites in the absence and presence of saccharides.     

Analysis of calcium binding to alpha-lactalbumin using a fluorescent calcium indicator.

A sensitive and rapid assay of Ca2+ binding to proteins was developed, based on the competition of Ca2+ binding to the protein of interest and fluo-3, a fluorescent Ca2+ indicator. Ca2+ binding to fluo-3 and bovine alpha-lactalbumin was analyzed at ten different pH values and a range of Na+ and K+ concentrations. We demonstrate that the binding constants of alpha-lactalbumin, determined by means of the competition assay and using intrinsic protein fluorescence, are the same within experimental error. The dissociation constant of the alpha-lactalbumin--Ca2+ complex in 50 mM Hepes containing 150 mM Na+ at pH 7.4 and 25 degrees C, was found to be 123 +/- 2 nM and 103 +/- 43 nM when determined by the competition assay and intrinsic protein fluorescence, respectively. Binding of Ca2+ to alpha-lactalbumin did not depend on pH in the range 6.6-8.4 and was differently affected by Na+ and K+. EDTA-agarose, a chelating chromatography material, was synthesized and used to remove Ca2+ from buffer and protein solutions. The total concentration of Ca2+ in 50 mM Hepes, containing 150 mM Na+ at pH 7.4, was lowered to 119 +/- 13 nM and the number of Ca2+ bound/molecule alpha-lactalbumin was lowered to 0.069 +/- 0.006. No interaction between fluo-3 and alpha-lactalbumin could be discerned from spectral analysis and fluorescence anisotropy measurements.     

Characterization of calcium binding to brush-border membranes from rat duodenum.

The Ca2+-binding properties of isolated brush-border membranes at physiological ionic strength and pH were examined by rapid Millipore filtration. A comprehensive analysis of the binding data suggested the presence of two types of Ca2+-binding sites. The high-affinity sites, Ka = (6.3 +/- 3.3) X 10(5) M-1 (mean +/- S.E.M.), bound 0.8 +/- 0.1 nmol of Ca2+/mg of protein and the low-affinity sites, Ka = (2.8 +/- 0.3) X 10(2) M-1, bound 33 +/- 3.5 nmol of Ca2+/mg of protein. The high-affinity site exhibited a selectivity for Ca2+, since high concentrations of competing bivalent cations were required to inhibit Ca2+ binding. The relative effectiveness of the competing cations (1 and 10 mM) for the high-affinity site was Mn2+ approximately equal to Sr2+ greater than Ba2+ greater than Mg2+. Data from the pH studies, treatment of the membranes with carbodi-imide and extraction of phospholipids with aqueous acetone and NH3 provided evidence that the low-affinity sites were primarily phospholipids and the high-affinity sites were either phosphoprotein or protein with associated phospholipid. Two possible roles for the high-affinity binding sites are suggested. Either high-affinity Ca2+ binding is involved with specific enzyme activities or Ca2+ transport across the luminal membrane occurs via a Ca2+ channel which contains a high-affinity Ca2+-specific binding site that may regulate the intracellular Ca2+ concentration and gating of the channel.     

Ions binding to S100 proteins. I. Calcium- and zinc-binding properties of bovine brain S100 alpha alpha, S100a (alpha beta), and S100b (beta beta) protein: Zn2+ regulates Ca2+ binding on S100b protein

Flow dialysis measurements of calcium binding to bovine brain S100 alpha alpha, S100a (alpha beta), and S100b (beta beta) proteins in 20 mM Tris-HCl buffer at pH 7.5 and 8.3 revealed that S100 proteins bind specifically 4 Ca2+ eq/mol of protein dimer. The specific calcium-binding sites had, therefore, been assigned to typical amino acid sequences on the alpha and beta subunit. The protein affinity for calcium is much lower in the presence of magnesium and potassium. Potassium strongly antagonizes calcium binding on two calcium-binding sites responsible for most of the Ca2+-induced conformational changes on S100 proteins (probably site II alpha and site II beta). Zinc-binding studies in the absence of divalent cations revealed eight zinc-binding sites/mol of S100b protein dimer that we assumed to correspond to 4 zinc-binding sites/beta subunit. Zinc binding to S100b studied with UV spectroscopy methods showed that the occupation of the four higher affinity sites and the four lower affinity sites on the protein dimer were responsible for different conformational changes in S100b structure. Zinc binding on the higher affinity sites regulates calcium binding to S100b by increasing the protein affinity for calcium and decreasing the antagonistic effect of potassium on calcium binding. Zinc-binding studies on S100a and S100 alpha alpha protein showed that the Trp-containing S100 proteins bind zinc more weakly than S100b protein. Calcium-binding studies on zinc-bound S100a proved that calcium- and zinc-binding sites were distinct although there was no increase in zinc-bound S100a affinity for calcium, as in S100b protein. Finally we provide evidence that discrepancies between previously published results on the optical properties of S100b protein probably result from oxidation of the sulfhydryl groups in the protein.     

Purification, characterization and ion binding properties of human brain S100b protein

Human brain S100b (beta beta) protein was purified using zinc-dependent affinity chromatography on phenyl-Sepharose. The calcium- and zinc-binding properties of the protein were studied by flow dialysis technique and the protein conformation both in the metal-free form and in the presence of Ca2+ or Zn2+ was investigated with ultraviolet spectroscopy, sulfhydryl reactivity and interaction with a hydrophobic fluorescence probe 6-(p-toluidino)naphthalene-2-sulfonic acid (TNS). Flow dialysis measurements of Ca2+ binding to human brain S100b (beta beta) protein revealed six Ca2+-binding sites which we assumed to represent three for each beta monomer, characterized by the macroscopic association constants K1 = 0.44 X 10(5) M-1; K2 = 0.1 X 10(5) M-1 and K3 = 0.08 X 10(5) M-1. In the presence of 120 mM KCl, the affinity of the protein for calcium is drastically reduced. Zinc-binding studies on human S100b protein showed that the protein bound two zinc ions per beta monomer, with macroscopic constants K1 = 4.47 X 10(7) M-1 and K2 = 0.1 X 10(7) M-1. Most of the Zn2+-induced conformational changes occurred after the binding of two zinc ions per mole of S100b protein. These results differ significantly from those for bovine protein and cast doubt on the conservation of the S100 structure during evolution. When calcium binding was studied in the presence of zinc, we noted an increase in the affinity of the protein for calcium, K1 = 4.4 X 10(5) M-1; K2 = 0.57 X 10(5) M-1; K3 = 0.023 X 10(5) M-1. These results indicated that the Ca2+- and Zn2+-binding sites on S100b protein are different and suggest that Zn2+ may regulate Ca2+ binding by increasing the affinity of the protein for calcium.     

Metal binding and metal-induced conformational change of frog bone gamma-carboxyglutamic acid-containing protein

Ca2+ or Cd2+ binding and the conformational change induced by the metal binding in two frog bone Gla-proteins (BGP, termed BGP-1 and BGP-2) were studied by equilibrium dialysis and CD measurement. By CD measurement in the far-ultraviolet region, the alpha-helix content of both apoBGPs was found to be 8%. Binding of both Ca2+ and Cd2+ was accompanied with a change in the CD spectrum, and the alpha-helix content increased to 15 and 25% for BGP-1 and BGP-2, respectively. CD measurement in the near-ultraviolet region indicated that the environment of aromatic amino acid residues in the protein molecule was changed by metal binding. Equilibrium dialysis experiments indicated that each of these two protein binds specifically 2 mol of Ca2+, and nonspecifically an additional 3-4 mol of Ca2+ in 0.02 M Tris-HCl/0.15 M NaCl (pH 7.4), at 4 degrees C. According to the two separate binding sites model, BGP-1 has 1 high-affinity Ca2+ binding site (Kd1 = 0.17 mM) and 1 low-affinity site (Kd2 = 0.29 mM), and BGP-2 contains 1 high-affinity site (Kd1 = 0.14 mM) and 1 low-affinity site (Kd2 = 0.67 mM). In addition, 2 Cd2+ bound to a high-affinity binding site on BGP-1 with Kd1 of 10.4 microM, and 1 Cd2+ bound to a low-affinity binding site with Kd2 of 41.5 microM. On the other hand, BGP-2 had three classes of binding sites and 1 Cd2+ bound to each binding site with Kd1 = 3.6 microM, Kd2 = 16.3 microM, Kd3 = 51.7 microM, respectively.(ABSTRACT TRUNCATED AT 250 WORDS)     

Heat capacity and entropy changes of the major isotype of the toad (Bufo) parvalbumin induced by calcium binding

The possible structural changes in the major isotype of parvalbumin from the toad (Bufo bufo japonicus) skeletal muscle caused by Ca2+ and Mg2+ binding have been analyzed by microcalorimetric titrations. Parvalbumin was titrated with Ca2+ in both the absence and presence of Mg2+ and with Mg2+ in the absence of Ca2+, at pH 7.0, and at 5 degrees, 15 degrees, and 25 degrees C. The two sites in a molecule were equivalent on Mg2(+)-Ca2+ exchange, but distinguishable on Ca2+ and Mg2+ binding. The reactions of parvalbumin with Ca2+ are exothermic at every temperature in both the absence and presence of Mg2+, but those with Mg2+ are always endothermic except for the binding to site 1 at 25 degrees C. The magnitudes of the hydrophobic and internal vibrational contributions to the heat capacity and entropy changes of parvalbumin on Ca2+ and Mg2+ binding and Mg2(+)-Ca2+ exchange have been estimated by the empirical method of Sturtevant [Sturtevant, J. M. (1977) Proc. Natl Acad. Sci. USA 74, 2236-2240]. Although no major conformational changes were noted between Ca2(+)- and Mg2(+)-bound forms of toad parvalbumin, the conformational difference was larger in Ca2+ (or Mg2+) binding to site 1 than site 2. This may indicate that the metal-free form is much less stable than any form with Ca2+ (or Mg2+) bound at one site at least. On Mg2(+)-Ca2+ exchange, the vibrational as well as hydrophobic entropy is only slightly increased in a parallel manner. In contrast, on Ca2+ (or Mg2+) binding, the hydrophobic entropy increases but the vibrational entropy decreases; the former indicates the sequestering of nonpolar groups from the surface to the interior of a molecule, and the latter suggests that the overall structures are tightened on Ca2+ (or Mg2+) binding but loosened on Mg2(+)-Ca2+ exchange. Despite the clear distinctions in the thermodynamic features, the conformational changes of toad parvalbumin are essentially the same as those of the two isotypes of bullfrog parvalbumins on Ca2+ binding and Mg2(+)-Ca2+ exchange.     

Effect of ethanol on troponin calcium binding

The Chelex resin method was found to be suitable for studying drug effects on Ca2+ binding of proteins. In comparison to conventional dialysis techniques, the Chelex method has the following advantages: Ca2+-EGTA buffer is not necessary, free Ca2+ concentration as low as 10(-9) M can be determined directly, and the reaction is complete in 30 min, thus creating fewer problems with protein denaturation at elevated temperatures. Methods to cope with problems inherent to this assay, such as the excluded volume effect of the resin and protein adsorption by the resin are described. The validity of the method was confirmed by the measurements of Ca2+ binding of troponin in the presence and absence of Mg2+. Using this method, it was demonstrated that ethanol concentration as high as 25% does not influence the Ca2+ binding of troponin.     

A calcium-43 NMR study of calcium binding to an acidic proline-rich phosphoprotein from human saliva

The 43Ca NMR line width measured for Ca2+ bound to protein A, an acidic proline-rich salivary protein, is 1 order of magnitude narrower than has previously been observed for other proteins of similar molecular weight. The correlation times, quadrupole coupling constants, and chemical shifts estimated for Ca2+ ions bound to the intact protein (Mr approximately 10 000) and its 30 amino acid residue long acidic N-terminal TX peptide were indistinguishable within experimental error. These results--as well as the outcome of 1H NMR relaxation rate measurements--are indicative of extensive motions for the protein residues, which in turn give rise to a high degree of flexibility for the protein-bound Ca2+. Ca2+ titration and pH-dependent measurements on protein A, the TX peptide, and the dephosphorylated TX peptide established the importance of the two phosphoserine residues in the binding of Ca2+. Moreover, a comparison of the 43Ca NMR parameters with those obtained for other Ca2+-binding proteins suggests the presence of Ca2+-binding sites of similar symmetry in all these proteins. No evidence was found for a proposed interaction between the highly acidic N-terminal and the weakly basic C-terminal regions of protein A. In contrast, the high pH inflection that was observed in the pH titration curve for the intact protein was also found for the phospho and dephospho TX peptides, thus suggesting that basic moieties in the N-terminal region rather than those in the C-terminal region may be responsible for this observation.     

Interaction of calcium with bovine plasma protein C

The binding of 45Ca2+ to bovine plasma protein C (PC) and to activated bovine plasma protein C (APC) has been examined by equilibrium ultrafiltration at pH 7.4 and 25 degrees C. Under these conditions, PC possesses 16.0 plus or minus 2.0 equivalent Ca2+ binding sites, of average KD (8.7 plus or minus 1.5) x 10(-4) M, and APC contains 9.0 plus or minus 1.0 equivalent Ca2+ binding sites, with an average KD of (4.3 plus or minus 1.1) x 10(-4) M. Both Mn2+ and Sr2+ were capable of ready displacement of Ca2+ from a Ca2+-PC complex, while Mg2+ was less effective in this regard. The alpha-thrombin-catalyzed activation of PC was inhibited by the presence of Ca2+. A kinetic analysis of this effect demonstrated that it was, in large part, due to an increase in the Km of the reaction. Addition of other divalent cations, e.g. Mn2+, Sr2+, and Mg2+, in place of Ca2+ also resulted in inhibition of the alpha-thrombin-catalyzed activation of PC in a manner which paralleled their ability to displace Ca2+ from a Ca2+-PC complex. On the other hand, the activation of PC by the coagulant protein from Russell's Viper venom was augmented by the presence of Ca2+. Other divalent metal ions, such as Sr2+ and Mn2+, in the absence of Ca2+, also weakly stimulated this reaction. Mg2+ was without notable effect.     

Calcyclin is a calcium and zinc binding protein

Calcyclin, a cell cycle regulated protein, was recently purified from Ehrlich ascites tumour (EAT) cells and shown to be a calcium binding protein. Here we show that calcyclin monomer and dimer also bind zinc ions. Zinc binding sites seem to be different from calcium binding sites since: preincubation with Ca2+ lacks effect on the binding of Zn2+, and Ca2+ (but not Zn2+) increases tyrosine fluorescence intensity. Binding of Zn2+ reduces the extent of the conformational changes induced by Ca2+, and seems to affect Ca2(+)-binding. The data suggest that Ca2+ and Zn2+ might trigger the biological activity of calcyclin.     

Thermodynamics of the binding of Ca2+ to porcine pancreatic phospholipase A2

The binding of Ca2+ to porcine pancreatic phospholipase A2 was studied by batch microcalorimetry. Enthalpies of binding at 25 degrees C were determined as a function of Ca2+ concentration in buffered solutions at pH 8.0 using both the Tris-HCl and Hepes-NaOH buffer systems. The calorimetric results indicate that protons are released on calcium binding and that in addition to the binding of the active-site calcium, there appears to be weak binding of a second Ca2+. Results from potentiometric titrations indicate that this proton release on binding Ca2+ arises from a change in pK of a histidine(s) functional group. The thermodynamic functions delta G0, delta H0 and delta S0 for calcium binding to phospholipase A2 have been determined. These results are compared with literature data for Ca2+ complex formation with some small molecules and also the protein troponin-C.     

Manganese, calcium, and saccharide binding to concanavalin A, as studied by ultrafiltration

The binding of the ligands Mn2+, Ca2+, and methyl alpha-D-glucopyranoside to concanavalin A, purified as described (A.J. Sophianopoulos and J.A. Sophianopoulos (1981) Prep. Biochem. 11, 413-435), was studied by ultrafiltration in 0.2 M NaCl, pH 5.2 and pH 6.5 to 7, and at 23 to 25 degrees C. The association constant (Ka) of methyl alpha-D-glucopyranoside to concanavalin A was (2 +/- 0.2) X 10(3) M-1, both at pH 5.2 and 7. At pH 5.2 and in the absence of Ca2+, the Ka of Mn2+ to concanavalin A was (5 +/- 1) X 10(3) M-1, and in the presence of 1 mM Ca2+, the Ka was (9.1 +/- 2.1) X 10(5) M-1. At pH 6.5 Mn2+ bound to concanavalin A with a Ka of (7.3 +/- 1.8) X 10(5) M-1, and the binding affinity was virtually independent of the presence of Ca2+. Experiments of binding of 4-methylumbelliferyl alpha-D-mannopyranoside to concanavalin A indicated that at pH 5.2, binding of a single Mn2+ per concanavalin A monomer was sufficient to induce a fully active saccharide binding site. Ca2+ is not necessary for such activation, but rather it increases the affinity of concanavalin A for binding Mn2+.     

Heat capacity and entropy changes of the two major isotypes of bullfrog (Rana catesbeiana) parvalbumins induced by calcium binding

The possible structural changes of the two major isotypes (PA1 and PA2) of parvalbumins from bullfrog (Rana catesbeiana) skeletal muscle caused by Ca2+ binding have been analyzed by microcalorimetric titrations. Titrations of the parvalbumins with Ca2+ have been made in both the absence and presence of Mg2+ at pH 7.0 and at 5, 15, and 25 degrees C. The reactions of the parvalbumins with Ca2+ are exothermic in both the presence and absence of Mg2+ and at every temperature. But the contributions of enthalpy and entropy changes are variable; Mg2+-Ca2+ exchange on PA1 at 25 degrees C is driven almost entirely by a favorable enthalpy change, whereas Ca2+ binding to PA2 at 5 degrees C is driven for the most part by a favorable entropy change. The magnitudes of the hydrophobic and internal vibrational contributions to the heat capacity and entropy changes of the parvalbumins on Ca2+ binding and Mg2+-Ca2+ exchange have been estimated by the empirical method of Sturtevant [Sturtevant, J. M. (1977) Proc. Natl. Acad. Sci. U.S.A. 74, 2236-2240]. Although PA1 (beta) and PA2 (alpha) belong to genetically different lineages, the parvalbumins indicate very similar conformational changes to each other on both Ca2+ binding and Mg2+-Ca2+ exchange. On Mg2+-Ca2+ exchange, the vibrational as well as hydrophobic entropy is slightly increased in a parallel manner. In contrast, on Ca2+ binding, the hydrophobic entropy increases but the vibrational entropy decreases. The increase in the hydrophobic entropy indicates the sequestering of nonpolar groups from the surface to the interior of molecules, while the changes in the vibrational entropy suggest that the overall structures are tightened on Ca2+ binding but loosened on Mg2+-Ca2+ exchange.     

The thermodynamics of calcium binding to thermolysin

Calcium binding isotherms were determined for thermolysin in the range pH 5.6-10.5, and from 5 to 45 degrees C. An extensive statistical analysis of the binding data suggests that at least two of the four binding sites bind Ca2+ with complete positive cooperativity and independently of the other two. Nonlinear regression analysis of the binding data was used to calculate cooperative (K1) and independent (K2) binding constants for the four calcium sites. Thermodynamic parameters obtained from a van't Hoff analysis indicate that calcium binding to both cooperative and independent sites is an entropy-driven process. At pH 7.0, delta H1 = 90.4 kJ/mol; delta H2 = 97.5 kJ/mol; delta S1 = 456 J K-1 mol-1; delta S2 = 262 J K-1 mol-1. These results are compared to those obtained for other calcium-binding proteins. An analysis of the pH dependence of the calcium binding constants indicates that the binding of four protons at the cooperative site and one to two protons at the independent sites, modulates the calcium affinity. This confirms an earlier structural assignment of the double-site as the locus of the two cooperatively binding Ca2+. Calcium binding to thermolysin is enhanced in the presence of an active site directed inhibitor, suggesting that there may be positive cooperativity between substrate and calcium binding.     

Low-temperature studies of the sarcoplasmic reticulum calcium pump. Mechanisms of calcium binding

The mechanism of the sarcoplasmic reticulum Ca2+-ATPase was investigated at low temperatures (0 to -12 degrees C). Transient states of the enzyme were studied by two complementary techniques: intrinsic protein fluorescence and rapid filtration on Millipore filters. Intrinsic fluorescence was used to distinguish conformational states of the protein and to evaluate the rate of conversion between these states. Filtrations were used to measure the evolution of the active sites during the transition; the time resolution was 2-5 s. At sub-zero temperatures this time is shorter than the lifetime of most of the enzymatic states which have been detected. In this paper the mechanism of Ca2+ binding to the protein is investigated in the absence of nucleotides. Two basic experiments are described; (1) Kinetics of calcium binding and dissociation over a wide range of calcium concentration. (2) Kinetics of calcium exchange (45Ca2+ in equilibrium 40Ca2+) at constant concentration. The results obtained in the first series of experiments are consistent with a sequential binding to two interacting Ca2+ binding sites. Calcium ions have very fast access to a site with low apparent affinity (Kd approximately 25 microM). Occupation of this site induces a slow conformational change which increased its apparent affinity and reveals a second site of high apparent affinity. At equilibrium the two sites are not equivalent in terms of rate of exchange. Two different rates were detected k fast greater than 0.2 s-1, k slow approximately 0.015 s-1 at -10 degrees C. Removal of Ca2+ from the fast exchanging site by addition of EGTA accelerates the rate of release of the slow exchanging one. A model is proposed with two interacting Ca2+-binding sites. A set of parameters has been obtained which produces correctly the Ca2+-binding curve and the fluorescence level at equilibrium as well as the rate constants of the calcium-induced fluorescence changes over a very wide range of Ca2+ concentrations (0.02 to 150 microM). The non-equivalence of the two classes of site and the meaning of the initial low-affinity binding are discussed.