Cadmium-substituted skeletal troponin C metal binding investigations and sequence assignment of the cadmium-113 resonances

The binding of cadmium to the calcium binding subunit of skeletal troponin (STnC) has been reinvestigated using direct binding methods and fluorescent derivatives. These data provide straightforward explanations of the observed titration behavior in the 113Cd NMR (Ellis, P.D., Strang, P., and Potter, J.D. (1984) J. Biol. Chem. 259, 10348-10356). Further, fluorescent derivatives of skeletal troponin C provide an excellent means of establishing a sequence assignment for the resonances observed in the 113Cd NMR. The results of these experiments demonstrate that sites I and II, the Ca2+ regulatory sites, can be assigned to resonances at -108.5 and -101.5 ppm, respectively. Sites III and IV, the structural sites, are assigned to resonances -112.8 and -106.8 ppm, respectively. These data are discussed in terms of recent structural findings and speculations.     

The calcium and magnesium binding sites on cardiac troponin and their role in the regulation of myofibrillar adenosine triphosphatase

The cardiac troponin (Tn) complex, consisting of a Ca2+-binding subunit (TnC), an inhibitory subunit (TnI), and a tropomyosin-binding subunit (TnT), has been reconstituted from purified troponin subunits isolated from bovine heart muscle. The Ca2+-binding properties of cardiac Tn were determined by equilibrium dialysis using either EGTA or EDTA to regulate the free Ca2+ concentration. Cardiac Tn binds 3 mol Ca2+/mol and contains two Ca2+-binding sites with a binding constant of 3 X 10(8) M-1 and one binding site with a binding constant of 2 X 10(6) M-1. In the presence of 4 mM MgC12, the binding constant of the sites of higher affinity is reduced to 3 X 10(7) M-1, while Ca2+ binding to the site at the lower affinity is unaffected. The two high affinity Ca2+-binding sites of cardiac Tn are analogous to the two Ca2+-Mg2+ sites of skeletal Tn, while the single low affinity site is similar to the two Ca2+-specific sites of skeletal Tn (Potter, J. D., and Gergely, J. (1975) J. Biol. Chem. 250, 4625-5633). The Ca2+-binding properties of the complex of TnC and TnI (1:1 molar ratio) were similar to those of Tn. Cardiac TnC also binds 3 mol of Ca2+/mol and contains two sites with a binding constant of 1 X 10(7) M-1 and a single site with a binding constant of 2 X 10(5) M-1. Assuming competition between Mg2+ and Ca2+ for the high affinity sites of TnC and Tn, the binding constants for Mg2+ were 0.7 and 3.0 X 10(3) M-1, respectively. The Ca2+ dependence of cardiac myofibrillar ATPase activity was similar to that of an actomyosin preparation regulated by the reconstituted troponin complex. Comparison by the Ca2+-binding properties of cardiac Tn and the cardiac myofibrillar ATPase activity as a function of [Ca2+] and at millimolar [Mg2+] suggests that activation of the ATPase occurs over the same range of [Ca2+] where the Ca2+-specific site of cardiac Tn binds Ca2+.     

Effect of Ca2+ binding on troponin C. Changes in spin label mobility, extrinsic fluorescence, and sulfhydryl reactivity.

The Ca2+ binding component (TnC) of troponin has been selectively labeled with either a spin label, N-(1-oxyl-2,2,6,6-tetramethyl-4-piperidinyl) iodoacetamide, or with a fluorescent probe, S-mercuric-N-dansyl cysteine, presumably at its single cysteine residue (Cys-98) in order to probe the interactions of TnC with divalent metals and with other subunits of troponin. The modified protein has the same Ca2+ binding properties as native TnC (Potter, J. D., and Gergely, J. (1975) J. Biol. Chem. 250, 4628), viz. two Ca2+ binding sites at which Mg2+ appears to compete (Ca2+-Mg2+ sites, KCa = 2 X 10(7) M-1) and two sites at which Mg2+ does not compete (Ca2+-specific sites, KCa = 2 X 10(5) M-1). Either Ca2+ or Mg2+ alters the ESR spectrum of spin-labeled TnC in a manner that indicates a decrease in the mobility of the label, Ca2+ having a slightly greater effect. In systems containing both Ca2+ and Mg2+ the mobility of the spin label is identical with that in systems containing Ca2+ alone. The binding constants for Ca2+ and Mg2+ deduced from ESR spectral changes are 10(7) and 10(3) M-1, respectively, and the apparent affinity for Ca2+ decreases by about an order of magnitude on adding 2 mM Mg2+. Thus, the ESR spectral change is associated with binding of Ca2+ to one or both of the Ca2+-Mg2+ sites. Addition of Ca2+ to the binary complexes of spin-labeled TnC with either troponin T (TnT) or troponin I (TnI) produces greater reduction in the mobility of the spin label than in the case of spin-labeled TnC alone, and in the case of the complex with TnI the affinity for Ca2+ is increased by an order of magnitude. The fluorescence of dansyl (5-dimethylaminonaphthalene-1-sulfonyl)-labeled TnC is enhanced by Ca2+ binding to both high and low affinity sites with apparent binding constants of 2.6 X 10(7) M-1 and 2.9 X 10(5) M-1, respectively, calculated from the transition midpoints. The presence of 2 mM Mg2+, which produces no effect on dansyl fluorescence itself, in contrast to its effect on the spin label, shifts the high affinity constant to 2 X 10(6) M-1. Spectral changes produced by Ca2+ binding to the TnC-TnI complex furnish evidence that the affinity of TnC for Ca2+ is increased in the complex. The reactivity of Cys-98 to the labels and to 5,5'-dithiobis(2-nitrobenzoic acid) (Nbs2) is decreased by Ca2+ or Mg2+ both with native TnC and in 6 M urea. The reaction rate between Cys-98 and Nbs2 decreases to one-half the maximal value at a Ca2+ concentration that suggests binding to the Ca2+-Mg2+ sites. Formation of a binary complex between TnI and TnC reduces the rate of reaction, and there is a further reduction by Ca2+. The effect of Ca2+ takes place at concentrations that are 1 order of magnitude lower than in the case of TnC alone. These results suggest that the Ca2+ binding site adjacent to Cys-98 is one of the Ca2+-Mg2+ binding sites.     

Calcium- and magnesium-binding properties of oncomodulin. Direct binding studies and microcalorimetry

Ca2+ binding to the wild type recombinant oncomodulin was studied by equilibrium flow dialysis in the absence and presence of 1, 2, and 10 mM Mg2+. Direct Mg2(+)-binding experiments were carried out by the Hummel-Dryer gel filtration technique. These studies revealed that in the absence of Mg2+ oncomodulin binds two Ca2+ with KCa = 2.2 x 10(7) and 1.7 x 10(6) M-1, respectively. In the absence of Ca2+ the protein binds only one Mg2+ with KMg = 4.0 x 10(3) M-1.Mg2+ antagonizes Ca2+ binding at the high affinity site according to the rule of direct competition. Ca2+ binding to the low affinity site is only slightly affected by Mg2+, so that in the presence of 2-3 mM Mg2+ the two sites have apparently an equal affinity for Ca2+. Microcalorimetry showed that, in spite of the different affinities of the two Ca2(+)-binding sites, delta H0 for the binding of each Ca2+ is identical and exothermic for -18.9 kJ/site. It follows that the entropy gain upon binding of Ca2+ is +77.1 J K-1 site-1 for the high affinity Ca2(+)-Mg2+ site and +56.0 J K-1 site-1 for the low affinity Ca2(+)-specific site. Mg2+ binding is endothermic for +13 kJ/site with an entropy change of +111 J K-1 site-1. The thermodynamic characteristics of the Ca2(+)-Mg2+ site resemble most those of site II (the so-called EF domain) of toad alpha-parvalbumin. The characteristics of Ca2+ binding to the specific site (likely the CD domain) are different from those of the Ca2+ specific sites in troponin C and in calmodulin and suggest that in oncomodulin hydrophobic forces do not play a predominant role in the binding process at the specific site.     

13C and 113Cd NMR studies of the chelation of metal ions by the calcium binding protein parvalbumin

13C NMR spectra are presented for the calcium binding protein parvalbumin (pI 4.25) from carp muscle in several different metal bound forms: with Ca2+ in both the CD and EF calcium binding sites, with Cd2+ in both sites, with 113Cd2+ in both sites, and with 113Cd2+ in the CD site and Lu3+ in the EF site. The different metals differentially shift the 13C NMR resonances of the protein ligands involved in chelation of the metal ion. In addition, direct 13C-113Cd spin-spin coupling is observed which allows the assignment of protein carbonyl and carboxyl 13C NMR resonances to ligands directly interacting with the metal ions in the CD and EF binding sites. The displacement of 113Cd2+ from the EF site by Lu3+ further allows these resonances to be assigned to the CD or EF site. The occupancy of the two sites in the two cadmium species and in the mixed Cd2+/Lu3+ species is verified by 113Cd NMR. The resolution in these 113Cd NMR spectra is sufficient to demonstrate direct interaction between the two metal binding sites.     

Resolution and calcium-binding properties of the two major isoforms of troponin C from crayfish

Crayfish tail muscle troponin C (TnC) has been fractionated into its five components and the Ca2+-binding properties of the two major isoforms (alpha and gamma) determined by equilibrium dialysis. alpha-TnC contains one Ca2+-binding site with a binding constant of 1 x 10(6) M-1 and one Ca2+ site with a binding constant of 1 x 10(4) M-1. In the complex of alpha-TnC with troponin I (TnI) or with TnI and troponin T (TnT), both sites bind Ca2+ with a single affinity constant of 2-4 x 10(6) M-1. gamma-TnC contains two Ca2+-binding sites with a binding constant of 2 x 10(4) M-1. In the gamma-TnC.TnI and gamma-TnC.TnI.TnT complexes, the binding constant of one of the sites is increased to 4-5 x 10(6) M-1, while Ca2+ binding to the second site is hardly affected (KCa = 4-7 x 10(4) M-1). In the presence of 10 mM MgCl2, the two Ca2+-binding sites of both TnC isoforms exhibit a 2-3-fold lower affinity. Assuming competition between Ca2+ and Mg2+ for these sites, their binding constants for Mg2+ were 120-230 M-1. In the absence of Ca2+, however, alpha-TnC and gamma-TnC bind 4-5 mol of Mg2+/mol with a binding constant of 1 x 10(3) M-1. These results suggest that the effect of Mg2+ on Ca2+ binding at the two Ca2+ sites is noncompetitive, i.e. Mg2+ does not bind directly to these sites (Ca2+-specific sites). Since the formation of the complex of crayfish TnI with alpha-TnC or gamma-TnC increases significantly the affinity of one of their two Ca2+-specific sites, I conclude that the binding of Ca2+ to only one site (regulatory Ca2+-specific site) controls the Ca2+-dependent interaction between crayfish TnCs and TnI.     

113Cd NMR study of bovine prothrombin fragment 1 and factor X

The interaction of Cd2+ with bovine prothrombin fragment 1, prothrombin intermediate 1, factor X, and a modified (Gla-domainless) factor X has been studied with 113Cd NMR. All the 113Cd resonances observed in this study were in the chemical shift range expected for oxygen ligands, suggesting that cadmium is binding at the same sites where calcium binds. Both fragment 1 and factor X displayed two major resonances, one near 10 ppm from 113Cd2+ that did not exchange rapidly with unbound 113Cd2+ (the high-affinity, or H, resonance) and one near -15 ppm from 113Cd2+ that exchanged rapidly with unbound 113Cd2+ (the low-affinity, or L, resonance). The difference between the chemical shift of the H resonance and the chemical shift range of -90 to -125 ppm that has been reported for three other small calcium-binding proteins is postulated to be due to different coordination geometries for monocarboxylate and dicarboxylate ligands; Cd2+ binds to fragment 1 and factor X through the dicarboxylate side chains of gamma-carboxyglutamate (Gla) residues. This allows contribution of only one oxygen per carboxyl group. At least one of the first few 113Cd2+ ions bound to fragment 1 did not appear in the 113Cd NMR spectrum until a total of five 113Cd2+ had been added. This could be due to exchange broadening of initial 113Cd2+ resonances due to sharing of ligands among several sites. Filling all sites would then restrict ligand exchange. Addition of Zn2+ displaced 113Cd2+ from the H resonance sites. Factor X did not display the interactions among ion binding sites proposed for fragment 1.     

Nuclear magnetic resonance investigation of cadmium 113 substituted pea and lentil lectins

The lentil (LcH) and pea (PSA) lectins, which are members of the class of D-glucose/D-mannose binding lectins, are Ca2+ X Mn2+ metalloproteins that require the metal ions for their saccharide binding and biological activities. We have prepared a variety of Cd2+ derivatives of PSA and LcH, with Cd2+ in either the transition metal (S1) or calcium (S2) sites, or in both. Thus, Cd2+ X Zn2+, Cd2+ X Mn2+, and Ca2+ X Cd2+ derivatives were prepared, in addition to the Cd2+ X Cd2+ derivatives which we have recently reported. This is the first report of stable mixed metal Cd2+ complexes of lectins. The physical and saccharide binding properties of the Cd2+ derivatives of both lectins were characterized by a variety of physiochemical techniques and found to be the same as those of the corresponding native proteins. 113Cd NMR spectra of mono- and disubstituted 113Cd2+ complexes of LcH and PSA were recorded and compared with 113Cd NMR data for concanavalin A (ConA) (Palmer, A.R., Bailey, D.B., Behnke, W.D., Cardin, A.D., Yang, P.P., and Ellis, P.D. (1980) Biochemistry 19, 5063-5070). The data for the PSA and LcH derivatives were found to be very similar, indicating close homology of their metal ion binding sites. 113Cd resonances at 44.6 ppm and -129.4 ppm for 113Cd2+ X 113Cd2+ X LcH, and at 46.6 and -130.4 for the corresponding PSA derivative, are chemical shifts very similar to those observed for 113Cd2+ X 113Cd2+ X ConA. Assignment of the resonances to the transition metal (S1) and calcium (S2) sites were unambiguous since the Ca2+ X 113Cd2+ and 113Cd2+ X Zn2+ derivatives of both lectins showed single resonances characteristic of the S1 and S2 sites, respectively. The results indicate that, unlike ConA, 113Cd2+ binds tightly to PSA and LcH. Binding of monosaccharide to both lectins induce small (2 ppm) upfield shifts in their S2 113Cd resonances, in contrast to the larger shift (8 ppm) observed in ConA. The 113Cd2+ X Mn2+ complexes of PSA and LcH fail to show a 113Cd resonance characteristic of these derivatives, which provides evidence for the close proximity of the metal ions in the two proteins. The present findings indicate that the coordinating ligand atoms to the metal ions at the S1 and S2 sites in LcH, PSA, and ConA are the same.     

Evidence for four capital and six auxiliary cation-binding sites on calmodulin: divalent cation interactions monitored by direct binding and microcalorimetry

Recently, Mills and Johnson [7] and our group [9] provided evidence that calmodulin contains, in addition to the four Ca2+-binding sites (capital sites), which are essential for drug- and enzyme-binding, a number of divalent cation-binding sites of different ion selectivity (auxiliary sites), which modulate drug-binding as well as the affinity of Ca2+ for the capital sites. In the present study, the number of auxiliary sites and their relationship to the capital sites were determined by equilibrium gel filtration and by flow microcalorimetry with Zn2+ and Mn2+ as selective probes for the auxiliary sites and with Cd2+ as a probe for both types of sites. In the absence of other divalent cations, 6 mol of Zn2+ bind to calmodulin with an identical affinity constant of 2,850 M-1 and a delta H0 of 106 kJ/mol calmodulin. In the presence of millimolar free Ca2+ calmodulin binds, in addition to four Ca2+, six Zn2+ with an affinity constant of 1,200 M-1 and a delta H0 of 47 kJ/mol calmodulin. The Zn2+-Ca2+ antagonism is governed by negative free energy coupling between the capital and auxiliary sites. In contrast, the Zn2+-Mg2+ antagonism follows the rule of straight competition at all six auxiliary sites. Mn2+ also binds exclusively to the auxiliary sites with affinity constants of 800 or 280 M-1 and delta H0 of 45 or 46 kJ/mol calmodulin in the absence and presence of saturating [Ca2+], respectively. Cd2+ binds to the capital sites with an affinity constant of 3.4 10(4) M-1 (delta H = 35 kJ/mol calmodulin) and to the auxiliary sites with ca. 100-fold lower affinity. The Zn2+ much greater than Mn2+ greater than or equal to Cd2+ greater than Mg2+ selectivity of the auxiliary sites corroborates the potencies of these cations in modulating drug binding. The auxiliary site-specific cations are unable to promote high-affinity complex formation between calmodulin and melittin.     

The calcium and magnesium binding sites on troponin and their role in the regulation of myofibrillar adenosine triphosphatase.

Purified troponin (Tn), the complex of the Ca-2+ binding subunit (TnC), the inhibitory subunit (TnI), and the tropomyosin binding subunit (TnT) binds 4 mol of Ca-2+ per mol. Two sites bind Ca-2+ with a binding constant of 5 times 10-8 M- minus 1, and two with a binding constant of 5 times 10-6 M- minus 1. In the presence of 2 mM MgCl2 the binding to four sites can be characterized with a single affinity constant of 5 times 10-6 M- minus 1. Purified TnC also binds 4 mol of Ca-2+ per mol; two sites have a binding constant of 2 times 10-7 M- minus 1 and two have one of 2 times 10-5 M- minus 1. In the presence of 2 mM MgCl2 the binding constant of the sites of higher affinity is reduced to 2 times 10-6 M- minus 1, while Ca-2+ binding to the sites of lower affinity is unaffected. Assuming competition between Mg-2+ and Ca-2+ for the high affinity sites on TnC and Tn, the changes in Ca-2+ binding can be accounted for with KMg values of 5 times 10-3 M- minus 1 and 5 times 10-4 M- minus 1, respectively. Tn and TnC bind 4 mol of Mg-2+ per mol in the absence of Cs-2+. The fact that at [Ca-2+] similar to 10- minus 5 M four Ca-2+ and only two Mg-2+ are bound per mol of TnC in the presence of 2 mM Mg-2+ further supports the view that there is direct competition between Mg-2+ and Ca-2+ for the high affinity Ca-2+ binding sites on TnC and Tn. These results then suggest that Tn and TnC contain six divalent cation binding sites: two high affinity Ca-2+ binding sites that also bind Mg-2+ competitively (Ca-2+-Mg-2+ sites); two sites with lower affinity for Ca-2+ that do not bind Mg-2+ (Ca-2+-specific sites); and two sites that bind Mg-2+ but not Ca-2+ (Mg-2+-specific sites). The complex of TnC and TnI (1:1 molar ratio) has the same binding properties as Tn, suggesting a conformational change in TnC upon interaction with TnI. Studies on myofibrillar ATPase activity as a function of free Ca-2+ concentration at two different free Mg-2+ concentrations suggest that full activation by Ca-2+ occurs only upon binding of Ca-2+ to the two Ca-2+-specific binding sites in Tn but does not require binding of Ca-2+ to the Ca-2+-Mg-2+ sites.     

Calcium binding to troponin C and troponin: effects of Mg2+, ionic strength and pH

Calcium binding to troponin C and troponin was examined by a metallochromic indicator method under various conditions to obtain a further understanding of the regulatory roles of these proteins in muscle contraction. Troponin C has four Ca binding sites, of which 2 sites have a high affinity of 4.5 X 10(6) M-1 for Ca2+ and the other 2 sites have a low affinity of 6.4 X 10(4) M-1 in a reaction medium consisting of 100 mM KCl, 20 mM MOPS-KOH pH 6.80 and 0.13 mM tetramethylmurexide at 20 degrees C. Magnesium also binds competitively to both the high and low affinity sites: the apparent binding constants are 1,000 M-1 and 520 M-1, respectively. Contrary to the claim by Potter and Gergely (J. Biol. Chem. 250, 4628-4633, 1975), the low affinity sites are not specific only for Ca2+. The high and low affinity sites of troponin C showed different dependence on the ionic strength: the high affinity sites were similar to GEDTA, while the low affinity sites were similar to calmodulin, which has a steeper ionic strength dependence than GEDTA. Ca binding to troponin C was not affected by change of pH between 6.5 and 7.2. Troponin I enhanced the apparent affinity of troponin C for Ca2+ to a value similar to that for troponin. Trifluoperazine also increased Ca binding to troponin C. Troponin has four Ca binding sites as does troponin C, but the affinities are so high that the precise analysis was difficult by this method. The apparent binding constants for Ca2+ and Mg2+ were determined to be 3.5 X 10(6) M-1 and 440 M-1, respectively, for low affinity sites under the same conditions as for troponin C, being independent of change in pH between 6.5 and 7.2. The competitive binding of Mg2+ to the low affinity sites of troponin is consistent with the results of Kohama (J. Biochem. 88, 591-599, 1980). The estimate for the high affinity sites is compatible with the reported results.     

Calcium-binding properties of troponin C in detergent-skinned heart muscle fibers

In order to obtain information with regard to behavior of the Ca2+ receptor, troponin C (TnC), in intact myofilament lattice of cardiac muscle, we investigated Ca2+-binding properties of canine ventricular muscle fibers skinned with Triton X-100. Analysis of equilibrium Ca2+-binding data of the skinned fibers in ATP-free solutions suggested that there were two distinct classes of binding sites which were saturated over the physiological range of negative logarithm of free calcium concentration (pCa): class I (KCa = 7.4 X 10(7) M-1, KMg = 0.9 X 10(3) M-1) and class II (KCa = 1.2 X 10(6) M-1, KMg = 1.1 X 10(2) M-1). The class I and II were considered equivalent, respectively, to the Ca2+-Mg2+ and Ca2+-specific sites of TnC. The assignments were supported by TnC content of the skinned fibers determined by electrophoresis and 45Ca autoradiograph of electroblotted fiber proteins. Dissociation of rigor complexes by ATP caused a downward shift of the binding curve between pCa 7 and 5, an effect which could be largely accounted for by lowering of KCa of the class II sites. When Ca2+ binding and isometric force were measured simultaneously, it was found that the threshold pCa for activation corresponds to the range of pCa where class II sites started to bind Ca2+ significantly. We concluded that the low affinity site of cardiac TnC plays a key role in Ca2+ regulation of contraction under physiological conditions, just as it does in the regulation of actomyosin ATPase. Study of kinetics of 45Ca washout from skinned fibers and myofibrils revealed that cardiac TnC in myofibrils contains Ca2+-binding sites whose off-rate constant for Ca2+ is significantly lower than the Ca2+ off-rate constant hitherto documented for the divalent ion-binding sites of either cardiac/slow muscle TnC or fast skeletal TnC.     

113Cd nuclear magnetic resonance of Cd(II) alkaline phosphatases

113Cd NMR spectra of 113Cd(II)-substituted Escherichia coli alkaline phosphatase have been recorded over a range of pH values, levels of metal site occupancy, and states of phosphorylation. Under all conditions resonances attributable to cadmium specifically bound at one or more of the three pairs of metal-binding sites (A, B, and C sites) are detected. By following changes in both the 113Cd and 31P NMR spectra of 113Cd(II)2 alkaline phosphatase during and after phosphorylation, it has been possible to assign the cadmium resonance that occurs between 140 and 170 ppm to Cd(II) bound to the A or catalytic site of the enzyme and the resonance occurring between 51 and 76 ppm to Cd(II) bound to B site, which from x-ray data is located 3.9 A from the A site. The kinetics of phosphorylation show that cadmium migration from the A site of one subunit to the B site of the second subunit follows and is a consequence of phosphate binding, thus precluding the migration as a sufficient explanation for half-of-the-sites reactivity. Rather, there is evidence for subunit-subunit interaction rendering the phosphate binding sites inequivalent. Although one metal ion, at A site, is sufficient for phosphate binding and phosphorylation, the presence of a second metal ion at B site greatly enhances the rate of phosphorylation. In the absence of phosphate, occupation of the lower affinity B and C sites produces exchange broadening of the cadmium resonances. Phosphorylation abolishes this exchange modulation. Magnesium at high concentration broadens the resonances to the point of undetectability. The chemical shift of 113Cd(II) in both A and B sites (but not C site) is different depending on the state of the bound phosphate (whether covalently or noncovalently bound) and gives separate resonances for each form. Care must be taken in attributing the initial distribution of cadmium or phosphate in the reconstituted enzyme to that of the equilibrium species in samples reconstituted from apoenzyme. Both 113Cd NMR and 31P NMR show that some conformational changes consequent to metal ion or phosphate binding require several days before the final equilibrium species is formed.     

The effect of Mg2+ on the Ca2+ binding to troponin C in rabbit fast skeletal myofibrils.

The effect of Mg2+ on the Ca2+ binding to rabbit fast skeletal troponin C and the CA2+ dependence of myofibrillar ATPase activity was studied in the physiological state where troponin C was incorporated into myofibrils. The Ca2+ binding to troponin C in myofibrils was measured directly by 45Ca using the CDTA-treated myofibrils as previously reported (Morimoto, S. and Ohtsuki, I. (1989) J. Biochem. 105, 435-439). It was found that the Ca2+ binding to the low and high affinity sites of troponin C in myofibrils was affected by Mg2+ competitively and the Ca2(+)- and Mg2(+)-binding constants were 6.20 x 10(6) and 1.94 x 10(2) M-1, respectively, for the low affinity sites, and 1.58 x 10(8) and 1.33 x 10(3) M-1, respectively, for the high affinity sites. The Ca2+ dependence of myofibrillar ATPase was also affected by Mg2+, with the apparent Ca2(+)- and Mg2(+)-binding constants of 1.46 x 10(6) and 276 x 10(2) M-1, respectively, suggesting that the myofibrillar ATPase was modulated through a competitive action of Mg2+ on Ca2+ binding to the low affinity sites, though the Ca2+ binding to the low affinity sites was not simply related to the myofibrillar ATPase.     

Isolation, expression, and mutation of a rabbit skeletal muscle cDNA clone for troponin I. The role of the NH2 terminus of fast skeletal muscle troponin I in its biological activity.

A cDNA for rabbit fast skeletal muscle troponin I (TnI) was isolated and sequenced. The clone contains a coding sequence predicting a 182-amino-acid protein with a molecular mass of 21,162 daltons. The translated sequence is different from that reported by Wilkinson and Grand (Wilkinson, J. M., and Grand, R. J. A. (1978) Nature 271, 31-35) in that Arg-153, Asp-154, and Leu-155 must be inserted into their original sequence. Amino acid sequencing of adult rabbit TnI confirmed this result. In order to investigate the role of the NH2 terminus of TnI in its biological activity, we have expressed a recombinant deletion mutant (TnId57), which lacks residues 1-57, in a bacterial expression system. Both wild type TnI (WTnI) and TnId57 inhibited acto-S1-ATPase activity and this inhibition could be fully reversed by troponin C (TnC) in the presence of Ca2+. Additionally both WTnI and TnId57 bound to an actin affinity column. Thus, both inhibitory actin binding and Ca(2+)-dependent neutralization by TnC were retained in TnId57. TnC affinity chromatography was used to compare the binding of TnI and TnId57 to TnC. Using this method, two types of interaction between TnC and TnI were observed: 1) one which is metal independent (or structural) and 2) one dependent on Ca2+ or Mg2+ binding to the Ca(2+)-Mg2+ sites of TnC. The same experiments with TnId57 demonstrated that the type 1 interaction was weakened, and type 2 binding was lost. This method also revealed an interaction between TnC and TnI which is dependent upon Ca2+ binding to the Ca(2+)-specific sites of TnC and which is retained in TnId57. Taken together, these results suggest that the NH2 terminus of TnI may constitute a Ca(2+)-Mg(2+)-dependent interaction site between TnC and TnI and play, in part, a structural role in maintaining the stability of the troponin complex while the COOH terminus of TnI contains a Ca(2+)-specific site-dependent interaction site for TnC as well as the previously demonstrated Ca(2+)-sensitive inhibitory and actin binding activities.     

Calcium binds cooperatively to the regulatory sites of the cardiac thin filament

To investigate the relationship between thin filament Ca2+ binding and activation of the MgATPase rate of myosin subfragment 1, native cardiac thin filaments were isolated and characterized. Direct measurements of 45Ca binding to the thin filament were consistent with non-cooperative binding to two high affinity sites (Ka 7.3 +/- 0.8 x 10(6) M-1) and either cooperative or non-cooperative binding to one low affinity site (Ka 4 +/- 2 x 10(5) M-1) per troponin at 25 degrees C, 30 mM ionic strength, pH 7.06. Addition of a low concentration of myosin subfragment 1 to the native thin filaments produced a Ca2+-regulated MgATPase activity with Kapp (2.5 +/- 1.3 x 10(5) M-1), matching the low affinity Ca2+ site. The MgATPase rate was cooperatively activated by Ca2+ (Hill coefficient 1.8). To determine whether Ca2+ binding to the low affinity sites was cooperative, native thin filament troponin was exchanged with troponin labeled on troponin C with 2-(4'-iodoacetamidanilo)naphthalene-6-sulfonic acid. From the Ca2+-sensitive fluorescence of this complex, Ca2+ binding was cooperative with a Hill coefficient of 1.7-2.0. Using the troponin-exchanged thin filaments, myosin subfragment 1 MgATPase rate activation was also cooperative and closely proportional to Ca2+ thin filament binding. Reconstitution of the thin filament from its components raised the Ca2+ affinity by a factor of 2 (compared with native thin filaments) and incorporation of fluorescently modified troponin raised the Ca2+ affinity by another factor of 2. Stoichiometrically reconstituted thin filaments produced non-cooperative MgATPase rate activation, contrasting with cooperative activation with native thin filaments, troponin-exchanged thin filaments and thin filaments reconstituted with a stoichiometric excess of troponin. The Ca2+-induced fluorescence transition of stoichiometrically reconstituted thin filaments was non-cooperative. These results suggest that Ca2+ binds cooperatively to the regulatory sites of the cardiac thin filament, even in the absence of myosin, and even though cardiac troponin C has only one Ca2+-specific binding site. A theoretical model for these observations is described and related to the experimental data. Well-known interactions between neighboring troponin-tropomyosin complexes are the proposed source of cooperativity and also influence the overall Ka. The data indicate that Ca2+ is four times more likely to elongate a sequence of troponin-tropomyosin units already binding Ca2+ than to bind to a site interior to a sequence of units without Ca2+.     

Calcium-proton and calcium-magnesium antagonisms in calmodulin: microcalorimetric and potentiometric analyses

Microcalorimetry, pH potentiometry, and direct binding studies by equilibrium dialysis or gel filtration were performed to determine the thermodynamic functions delta Ho, delta Go, and delta So guiding the interactions of Ca2+, Mg2+, and H+ with bovine brain calmodulin. At pH 7.5, Ca2+ and Mg2+ binding are both endothermic with enthalpy changes of 19.5 and 72.8 kJ X (mol of calmodulin)-1, respectively. These enthalpy changes are identical for each of the four ion-binding domains. The affinity constants also are identical with intrinsic values of 10(5) M-1 for Ca2+ and 140 M-1 for Mg2+. Ca2+ and Mg2+ do not compete for the same binding sites: at high concentrations of both ions, a calmodulin-Ca4-Mg4 species is formed with an enthalpy value of 24.4 kJ X mol-1 with respect to calmodulin-Ca4 and -28.8 kJ X mol-1 with respect to calmodulin-Mg4. Moreover, in the presence of high concentrations of Ca2+, the affinity of each of the four ion-binding domains in calmodulin for Mg2+ is decreased by a factor of 4 and vice versa, indicative of negative free-energy coupling between Ca2+ and Mg2+ binding. Protons antagonize Ca2+ and Mg2+ binding in a different manner. Ca2+-H+ antagonism is identical in each of the four Ca2+-binding domains in the pH range 7.5-5.2. Our analyses suggest that three chemical geometries, probably carboxyl-carboxylate interactions, are responsible for this antagonism with ionization constants of 10(6.2) M-1 in the metal-free protein. Mg2+-H+ antagonism also is identical for each of the Mg2+-binding sites but is qualitatively different from Ca2+-H+ antagonism.(ABSTRACT TRUNCATED AT 250 WORDS)     

Luminescence studies of Tb3+ bound to the high affinity sites of the Ca2+-ATPase of sarcoplasmic reticulum

Direct excitation of lanthanide luminescence with a pulsed dye laser has been used to probe the molecular environment of the high affinity sites of the sarcoplasmic reticulum Ca2+-ATPase. The direct excitation spectrum of Tb3+ bound to these sites has been determined and a luminescence lifetime of approximately 1 ms measured. Measurements of the difference in lifetime of the Tb X ATPase complex in H2O and D2O indicate that there are approximately 2 H2O molecules in the first coordination sphere of Tb3+ bound at the high affinity sites of the ATPase. The results are compared with the properties of Tb3+ binding to high affinity sites of other Ca2+ binding proteins. The binding constant of Tb3+ to the ATPase is in the range of 0.3-5.0 X 10(8) M-1 as inferred from the KI for inhibition of ATP hydrolysis, in agreement with a previous report (Highsmith, S. R., and Head, M. R. (1983) J. Biol. Chem. 258, 6858-6862). The values of the Ca2+ binding constant (approximately 2 X 10(6) M-1) and the cooperative nature (n = 1.9) of Ca2+ protection of Tb3+ inhibition indicate that Tb3+ and Ca2+ compete for the high affinity sites of the ATPase. The results demonstrate that directly-excited Tb3+ luminescence provides unique information on the environment of the Ca2+ binding-transport sites of the SR ATPase.     

Kinetics of Ca2+ release shows interactions between the two classes of sites of troponin-C

The kinetics of Ca2+-release from the two high affinity sites of troponin-C (TnC) was studied by the stopped flow technique following rapid mixing with either EDTA or excess TbCl3. The rate constants obtained by the two methods were 2.8 and 0.7 s-1, respectively. For the tryptic fragment of TnC that contains only the COOH-terminal half of the molecule, both methods generate rate constants of 2.2 s-1. These results are consistent with the interpretation that binding of Tb3+ to the Ca2+-specific sites reduces the rate of dissociation of Ca2+ from, and thereby enhances the affinity for, the Ca2+-Mg2+ sites; this, in turn, suggests interactions between the two halves of the TnC molecule.     

Ca(2+)-dependence of structural changes in troponin-C in demembranated fibers of rabbit psoas muscle.

The Ca(2+)-dependence of structural changes in troponin-C (TnC) has been detected by monitoring the fluorescence from TnC labeled at Methionine-25, in the NH2-terminal domain, with danzylaziridine (TnC-DANZ) and then exchanged for endogenous TnC in glycerinated single fibers. The fluorescence-pCa relation obtained from fibers stretched to a sarcomere length greater than 4.0 microns evidenced two transitions: a small one, attributable to the binding of Ca2+ to the high affinity, Ca(2+)-Mg(2+)-binding sites of TnC; and a large one, attributable to the binding of Ca2+ to the low affinity, Ca(2+)-specific binding sites of TnC. In the fluorescence-pCa relation determined with fibers set to a sarcomere length of 2.4 microns, hence obtained in the presence of cycling cross-bridges, the large transition had the same Ca(2+)-dependence as did the development of tension. These results indicate that the NH2-terminal globular domain of TnC is modified by the binding of Ca2+ to sites located in both globular domains and that the structural changes in TnC resulting from the binding of Ca2+ to the low-affinity sites, but not to the high-affinity sites, are directly associated with the triggering of contraction.