Cooperative Effect of Calcium Binding to Adjacent Troponin Molecules on the Thin Filament-Myosin Subfragment 1 MgATPase Rate
1997; Elsevier BV; Volume: 272; Issue: 20 Linguagem: Inglês
10.1074/jbc.272.20.13196
ISSN1083-351X
AutoresCarol Butters, Jeremy Tobacman, Larry S. Tobacman,
Tópico(s)Cardiovascular Effects of Exercise
ResumoThe myosin subfragment 1 (S1) MgATPase rate was measured using thin filaments with known extents of Ca2+ binding controlled by varying the ratio of native cardiac troponin versus an inhibitory troponin with a mutation in the sole regulatory Ca2+ binding site of troponin C. Fractional MgATPase activation was less than the fraction of troponins that bound Ca2+, implying a cooperative effect of bound Ca2+ on cross-bridge cycling. Addition of phalloidin did not alter cooperative effects between bound Ca2+ molecules in the presence or absence of myosin S1. When the myosin S1 concentration was raised sufficiently to introduce cooperative myosin-myosin effects, lower Ca2+concentrations were needed to activate the MgATPase rate. MgATPase activation remained less than Ca2+ binding, implying a true, not just an apparent, increase in Ca2+ affinity. MgATPase activation by Ca2+ was more cooperative than could be explained by cooperativeness of overall Ca2+binding, the discrepancy between Ca2+ binding and MgATPase activation, or interactions between myosins. The results suggest the thin filament-myosin S1 MgATPase cycle requires calcium binding to adjacent troponin molecules and that this binding is cooperatively promoted by a single cycling cross-bridge. This mechanism is a potential explanation for Ca2+-mediated regulation of cross-bridge kinetics in muscle fibers. The myosin subfragment 1 (S1) MgATPase rate was measured using thin filaments with known extents of Ca2+ binding controlled by varying the ratio of native cardiac troponin versus an inhibitory troponin with a mutation in the sole regulatory Ca2+ binding site of troponin C. Fractional MgATPase activation was less than the fraction of troponins that bound Ca2+, implying a cooperative effect of bound Ca2+ on cross-bridge cycling. Addition of phalloidin did not alter cooperative effects between bound Ca2+ molecules in the presence or absence of myosin S1. When the myosin S1 concentration was raised sufficiently to introduce cooperative myosin-myosin effects, lower Ca2+concentrations were needed to activate the MgATPase rate. MgATPase activation remained less than Ca2+ binding, implying a true, not just an apparent, increase in Ca2+ affinity. MgATPase activation by Ca2+ was more cooperative than could be explained by cooperativeness of overall Ca2+binding, the discrepancy between Ca2+ binding and MgATPase activation, or interactions between myosins. The results suggest the thin filament-myosin S1 MgATPase cycle requires calcium binding to adjacent troponin molecules and that this binding is cooperatively promoted by a single cycling cross-bridge. This mechanism is a potential explanation for Ca2+-mediated regulation of cross-bridge kinetics in muscle fibers. Just as isometric tension is cooperatively activated by Ca2+, so is the cardiac thin filament-myosin S1 1The abbreviations used are: myosin S1, myosin subfragment 1; TnC, TnT, TnI, troponin C, T, and I, respectively; CBMII, mouse troponin C mutant D65A/E66A; CBMII-Tn, troponin reconstituted from CBMII. 1The abbreviations used are: myosin S1, myosin subfragment 1; TnC, TnT, TnI, troponin C, T, and I, respectively; CBMII, mouse troponin C mutant D65A/E66A; CBMII-Tn, troponin reconstituted from CBMII. MgATPase rate, even under conditions where there is no cooperativity in myosin S1 binding (1Tobacman L.S. Biochemistry. 1987; 26: 492-497Crossref PubMed Scopus (15) Google Scholar, 2Walsh T.P. Trueblood C.E. Evans R. Weber A. J. Mol. Biol. 1984; 182: 265-269Crossref Scopus (34) Google Scholar). A possible explanation for this behavior is that ATPase activation is proportional to Ca2+ binding to the many TnCs on each thin filament and that this calcium binding is cooperative (3Tobacman L.S. Sawyer D. J. Biol. Chem. 1990; 265: 931-939Abstract Full Text PDF PubMed Google Scholar). We tested the idea that Ca2+ binding and MgATPase activation are proportional and found to the contrary that they are not. Instead, fractional MgATPase activation was considerably less than fractional Ca2+binding and more closely paralleled the number of pairs of adjacent troponins with Ca2+ bound to both. To accomplish the above experiment, we employ a constitutively inhibitory form of cardiac troponin containing an inactivating mutation of the sole regulatory site of TnC, site II (4Huynh Q. Butters C.A. Leiden J.M. Tobacman L.S. Biophys. J. 1996; 70: 1447-1455Abstract Full Text PDF PubMed Scopus (23) Google Scholar). This troponin, designated CBMII-Tn, results in a low thin filament-myosin S1 MgATPase rate that is not increased by the addition of Ca2+, analogous to previous results in which a similar TnC mutant was exchanged into myofibrils or muscle fibers (5Dotson D.G. Putkey J.A. J. Biol. Chem. 1993; 268: 24067-24073Abstract Full Text PDF PubMed Google Scholar, 6Negele J.C. Dotson D.G. Liu W. Sweeney H.L. Putkey J.A. J. Biol. Chem. 1992; 267: 825-831Abstract Full Text PDF PubMed Google Scholar, 7Putkey J.A. Sweeney H.L. Campbell S.T. J. Biol. Chem. 1989; 264: 12370-12378Abstract Full Text PDF PubMed Google Scholar). CBMII-Tn binds to actin-tropomyosin with an affinity identical to that of normal troponin in the absence of Ca2+. This binding, which is very tight for both normal troponin and for CBMII-Tn (4Huynh Q. Butters C.A. Leiden J.M. Tobacman L.S. Biophys. J. 1996; 70: 1447-1455Abstract Full Text PDF PubMed Scopus (23) Google Scholar, 8Dahiya R. Butters C.A. Tobacman L.S. J. Biol. Chem. 1994; 269: 29457-29461Abstract Full Text PDF PubMed Google Scholar, 9Fisher D. Wang G. Tobacman L.S. J. Biol. Chem. 1995; 270: 25455-25460Abstract Full Text Full Text PDF PubMed Scopus (44) Google Scholar), permits the present report in which thin filaments are assembled with defined mixtures of normal troponin and CBMII-Tn. In the presence of saturating Ca2+ concentrations, such thin filaments exhibit a fractional saturation of the TnC regulatory sites that is experimentally controllable by varying the relative concentrations of the two forms of troponin. This permits assessment of Ca2+-regulated myosin S1 MgATPase activity in a novel manner as a function of bound Ca2+ rather than as a function of the free Ca2+. In addition to varying the ratio of the two troponins, the myosin S1 and free Ca2+ concentrations are also systematically varied in the present study. The results imply a previously unrecognized aspect of the cooperativity of muscle activation, that rapid cycling of an isolated cross-bridge depends on Ca2+ binding to adjacent troponin molecules, and also suggest that cross-bridge cycling increases Ca2+ affinity at least locally, regardless of the density of myosin on the thin filament. The relationship between the data and various models of thin filament structure and regulation are discussed. Cardiac troponin and tropomyosin were purified from bovine heart using an ether powder technique (10Tobacman L.S. Adelstein R.S. Biochemistry. 1986; 25: 798-802Crossref PubMed Scopus (61) Google Scholar). Rabbit fast skeletal muscle F-actin was obtained by the method of Spudich and Watt (11Spudich J.A. Watt S. J. Biol. Chem. 1971; 246: 4866-4871Abstract Full Text PDF PubMed Google Scholar). Because bovine cardiac myosin S1 tends to precipitate at the concentrations used in many of the experiments, most of the data were obtained using rabbit fast skeletal muscle chymotryptic myosin S1 purified by ion exchange chromatography (12Weeds A.G. Taylor R.S. Nature. 1975; 257: 54-56Crossref PubMed Scopus (930) Google Scholar). Some of the experiments (see Fig. 1) were repeated using bovine cardiac myosin S1 purified as described previously (10Tobacman L.S. Adelstein R.S. Biochemistry. 1986; 25: 798-802Crossref PubMed Scopus (61) Google Scholar). CBMII-Tn was prepared by reconstitution (13Tobacman L.S. Lee R. J. Biol. Chem. 1987; 262: 4059-4064Abstract Full Text PDF PubMed Google Scholar) of the ternary troponin complex from bovine cardiac TnI and TnT (13Tobacman L.S. Lee R. J. Biol. Chem. 1987; 262: 4059-4064Abstract Full Text PDF PubMed Google Scholar) and recombinant murine TnC mutant CBMII (4Huynh Q. Butters C.A. Leiden J.M. Tobacman L.S. Biophys. J. 1996; 70: 1447-1455Abstract Full Text PDF PubMed Scopus (23) Google Scholar). F-actin, tropomyosin, and various mixtures of troponin and CBMII-Tn were combined in the indicated ratios under the ionic conditions used in the ATPase experiments. Since troponin binds to the thin filament with an affinity of 3–5 × 108m−1 (8Dahiya R. Butters C.A. Tobacman L.S. J. Biol. Chem. 1994; 269: 29457-29461Abstract Full Text PDF PubMed Google Scholar, 9Fisher D. Wang G. Tobacman L.S. J. Biol. Chem. 1995; 270: 25455-25460Abstract Full Text Full Text PDF PubMed Scopus (44) Google Scholar) and the μm amounts of the troponins included in the present experiments were stoichiometric or slightly sub-stoichiometric relative to the actin concentration, it was anticipated that essentially all of both added forms of troponin would be bound to the thin filament. This was tested by a sedimentation experiment. 15.5 μmF-actin, 2 μm tropomyosin, 1 μm cardiac troponin (nonradioactive), and 1 μm reconstituted CBMII-Tn labeled with iodo[14C]acetic acid on TnT Cys-39 were combined in the presence of 100 μmCaCl2, 5 mm Tris-HCl (pH 7.5), 3.5 mm MgCl2, 8 mm KCl, 1 mm dithiothreitol. The sample was sedimented in a TL100 centrifuge at 25 °C for 20 min at 35,000 rpm. Quantitative SDS-polyacrylamide gel electrophoresis analysis by gel scanning and standard curve comparison (14Cassell M. Tobacman L.S. J. Biol. Chem. 1996; 271: 12867-12872Abstract Full Text Full Text PDF PubMed Scopus (46) Google Scholar) and liquid scintillation counting of samples indicated sedimentation of 94% of both troponins combined (assessed by SDS-polyacrylamide gel electrophoresis) and 92% of the labeled troponin (assessed by radioactivity). The fraction of actin pelleting was similar, 93%. All three values agreed within experimental error. The ATPase rate was measured by the release of radioactive phosphate from [γ-32P]ATP (15Pollard T.D. Korn E.D. J. Biol. Chem. 1973; 248: 4682-4690Abstract Full Text PDF PubMed Google Scholar) (NENTM Life Science Products, 2–7 × 107cpm/μmol) with five or more time points obtained at variable intervals of 20, 60, or 120 s, depending upon the ATPase rate. Conditions and protein concentrations were varied as described in each figure. The free Ca2+ concentration was varied using 0.5 mm1,2-bis-(2-amino-5-bromo-phenoxy)ethane-N,N,N′,N′-tetraacetic acid as a Ca2+ chelator and variable concentrations of CaCl2 (3Tobacman L.S. Sawyer D. J. Biol. Chem. 1990; 265: 931-939Abstract Full Text PDF PubMed Google Scholar). This fraction is equivalent to the fraction of adjacent troponin·troponin pairs in contrast to the other possible adjacent pairs: CBMII-Tn·CBMII-Tn, troponin·CBMII-Tn, and CBMII-Tn·troponin. The number of such boundaries depends upon two factors: (i) the relative amounts of the two troponins and (ii) the tendency of the two forms of troponin to bind in a random or nonrandom pattern. The fractional Ca2+saturation is θ = troponin/(troponin + CBMII-Tn). If binding were random, then the fraction of adjacent pairs with Ca2+ bound on both elements of the pair would simply equal θ2. However, prior work shows that when the two forms of troponin are present in excess, they compete in a way that implies positive cooperativity and a small tendency for the two forms of troponin to segregate from each other (4Huynh Q. Butters C.A. Leiden J.M. Tobacman L.S. Biophys. J. 1996; 70: 1447-1455Abstract Full Text PDF PubMed Scopus (23) Google Scholar). This same tendency must be assumed to exist in the present experiments, which differ in that the troponins are added in a stoichiometric amount relative to the sites on the actin filament. For a closed linear filament including n troponins, withp = nθ designated as the number of troponins with bound Ca2+, the fraction of tropomyosin·tropomyosin boundaries with Ca2+ bound on both sides of the boundary can be shown to be as follows. f22=∑Y−j(p−j)pjn−p−1j−1n×∑Y−jpjn−p−1j−1Equation 1 The sums are taken over j, which refers to the number of discrete regions of one or more consecutive Ca2+ along the filament. By induction, j varies from 1 to pwhen p < n/2, and j varies from 1 to n − p when p >n/2 (exceptions are when p = 0 orp = n). Y is the cooperativity parameter, corresponding not only to the tendency for each type of troponin to segregate from the other (4Huynh Q. Butters C.A. Leiden J.M. Tobacman L.S. Biophys. J. 1996; 70: 1447-1455Abstract Full Text PDF PubMed Scopus (23) Google Scholar) but also, in the case of a fully normal thin filament, to a Y-fold tighter Ca2+ binding to a troponin that is adjacent to a troponin already with bound Ca2+ (3Tobacman L.S. Sawyer D. J. Biol. Chem. 1990; 265: 931-939Abstract Full Text PDF PubMed Google Scholar). Consequently, each configuration has a statistical weighting factor ofY −j, accounting for its free energy relative to configurations with different values for j. The number of adjacent, bound Ca2+·Ca2+ pairs equals p − j for any configuration. The other terms in the expression have to do with the number of ways of placing j regions containing pCa2+ions in n places. Simulations (not shown) with Equation 1 show it to be indistinguishable from f 22 = θ2 = (p/n)2 when Y = 1 as expected because binding is random when Y has this value. Also, Equation 1 gives negligibly different results forn = 30 and n = 200 unless Yis much larger than is true for the present experiments. Finally, Equation 1 is numerically indistinguishable from the implicit relationship between f 22 and θ that arises from independent derivations of the functionsf 22(Ca2+) and θ(Ca2+) (16Hill T.L. Cooperativity Theory in Biochemistry. Springer-Verlag New York Inc., New York1985Crossref Google Scholar). Fig. 1 shows the effect of altering the fractional Ca2+ saturation of the thin filament by varying the relative concentrations of troponin and CBMII-Tn. The normalized results of six experiments are shown, and it is clear that the relationship between Ca2+ binding and MgATPase rate activation is not a linear one (straight line). Rather, activation lags behind Ca2+ binding. When 50% of the troponins bind Ca2+ and 50% do not, the fractional MgATPase rate activation is only 30–35% that of the maximal stimulation observed for full Ca2+ saturation. Thesolid line, which does not fit the data, is the result expected if MgATPase activation were proportional to Ca2+binding regardless of whether Ca2+ binding is cooperative. The dashed lines in Fig. 1 are theoretical curves for the fraction of troponin·troponin boundaries with Ca2+ bound on both sides, which depends in part upon the degree of cooperativity in the binding of the two forms of troponin to the thin filament. The equilibrium constant Y governs the tendency of troponin and CBMII-Tn to separately cluster on the thin filament rather than bind randomly (3Tobacman L.S. Sawyer D. J. Biol. Chem. 1990; 265: 931-939Abstract Full Text PDF PubMed Google Scholar, 4Huynh Q. Butters C.A. Leiden J.M. Tobacman L.S. Biophys. J. 1996; 70: 1447-1455Abstract Full Text PDF PubMed Scopus (23) Google Scholar). Y also dictates the cooperativity of Ca2+ binding to a thin filament containing only normal troponin, with Y > 1 indicating positive cooperativity. The experimentally determined value ofY is approximately 1.5 (4Huynh Q. Butters C.A. Leiden J.M. Tobacman L.S. Biophys. J. 1996; 70: 1447-1455Abstract Full Text PDF PubMed Scopus (23) Google Scholar), and the long dashesin Fig. 1 correspond to this value. A slightly better fit is found withY = 4, as indicated by the theoretical curve represented with shorter dashes. This might suggest that the true value for Y is greater than the previously measured value of about 1.5. A more likely explanation is that the degree of MgATPase rate activation does not precisely correspond to the fraction of troponin·troponin pairs that have Ca2+ on both sides. In either case, the deviation from linearity in Fig. 1 indicates that Ca2+ binding to more than one troponin is required for full actin activation of ATP hydrolysis at any given thin filament site. An important aspect of the cooperative process illustrated in Fig. 1 is that it is not due to interactions between myosin S1 molecules. The myosin S1 concentration was only 1% that of the actin concentration, making myosin·myosin cooperativity unlikely. To confirm this experimentally, the MgATPase rate was shown to be linear with the myosin S1 concentration over a 16-fold range (0.25–4% that of the actin concentration). Linearity with myosin S1 concentration was shown both at pCa 5 and at pCa 5.89 (10–15% activation) for thin filaments with troponin and no CBMII-Tn and atpCa 5 for thin filaments with 50% troponin and 50% CBMII-Tn (data not shown). The curvature in Fig. 1 is not attributable to hyperbolic dependence of the MgATPase rate on the regulated actin concentration in the presence of saturating Ca2+ concentrations (10Tobacman L.S. Adelstein R.S. Biochemistry. 1986; 25: 798-802Crossref PubMed Scopus (61) Google Scholar, 17Adelstein R.S. Eisenberg E. Annu. Rev. Biochem. 1980; 49: 921-956Crossref PubMed Scopus (743) Google Scholar). Any such tendency would work in the reverse direction, producing a convex relationship or else tending to straighten a concave curve such as shown. This is not a major factor in Fig. 1 in any case because the MgATPase rates for the Ca2+-saturated thin filaments are about one-fourth to one-third the V max observed with saturating thin filament concentrations (data not shown). The actin concentration for the data sets in the figure are below the actinK app, which diminishes the importance of this consideration. The polymerization ability of the troponin·tropomyosin complex (18Jackson P. Amphlett G.N. Perry S.V. Biochem. J. 1975; 151: 85-97Crossref PubMed Scopus (63) Google Scholar, 19White S.P. Cohen C. Phillips Jr., G.N. Nature. 1987; 325: 826-828Crossref PubMed Scopus (180) Google Scholar, 20Phillips Jr., G.N. Fillers J.P. Cohen C. J. Mol. Biol. 1986; 192: 111-131Crossref PubMed Scopus (269) Google Scholar) and atomic models of actin·actin contacts in F-actin (4Huynh Q. Butters C.A. Leiden J.M. Tobacman L.S. Biophys. J. 1996; 70: 1447-1455Abstract Full Text PDF PubMed Scopus (23) Google Scholar, 21Lorenz M. Poole K.J.V. Popp D. Rosenbaum G. Holmes K.C. J. Mol. Biol. 1995; 246: 108-119Crossref PubMed Scopus (190) Google Scholar) suggest that longitudinal contacts along the thin filament are the most likely source of cooperativity. However, this does not exclude the possibility that cooperativity occurs across rather than along the actin filament. To test this, we added phalloidin, which binds near the thin filament axis with an orientation that is invariant with thin filament conformation (22Naber N. Ostap E.M. Thomas D.D. Cooke R. Proteins. 1993; 17: 347-354Crossref PubMed Scopus (6) Google Scholar) and both decreases thin filament flexibility and alters strand-strand interactions (23Orlova A. Prochniewicz E. Egelman E.H. J. Mol. Biol. 1995; 245: 598-607Crossref PubMed Scopus (146) Google Scholar, 24Lorenz M. Popp D. Holmes K.C. J. Mol. Biol. 1993; 234: 826-836Crossref PubMed Scopus (445) Google Scholar, 25Isambert H. Venier P. Maggs A.C. Fattoum A. Kassab R. Pantaloni D. Carlier M.-F. J. Biol. Chem. 1995; 270: 11437-11444Abstract Full Text Full Text PDF PubMed Scopus (471) Google Scholar). Any cooperativity that was dependent upon such interactions might be changed by the addition of phalloidin. The Fig. 2 inset shows that the cooperative effect of bound Ca2+ on MgATPase rate activation was similar to results found in the absence of phalloidin. The results are indistinguishable from Fig. 1. The main portion of Fig. 2 provides a measurement of Ca2+-dependent cooperative interactions between troponin molecules on the thin filament in the absence of myosin, again in the presence of phalloidin. This experiment differs from the ATPase data in that the sum of the troponin and CBMII-Tn concentrations is in constant excess relative to the sites on the thin filament. The two forms of troponin compete for binding sites on actin-tropomyosin, and the pattern of this competition implies that these binding sites (for troponin) interact in a manner sensitive to Ca2+. This experiment measures the value of the cooperativity parameter and equilibrium constant Y, which is found to be 1.7 ± 0.4 in the presence of phalloidin. This result implies weak Ca2+-sensitive interactions of a strength indistinguishable from that found previously in the absence of phalloidin (4Huynh Q. Butters C.A. Leiden J.M. Tobacman L.S. Biophys. J. 1996; 70: 1447-1455Abstract Full Text PDF PubMed Scopus (23) Google Scholar). Curve-fitting of the data also results in a value forK R, the fold-increase in the affinity of troponin for actin·tropomyosin that results from Ca2+dissociation from site II. K R is 2.4 ± 0.2 in the presence of phalloidin, which is indistinguishable fromK R in the absence of phalloidin, 2.2 ± 0.1 (4Huynh Q. Butters C.A. Leiden J.M. Tobacman L.S. Biophys. J. 1996; 70: 1447-1455Abstract Full Text PDF PubMed Scopus (23) Google Scholar). The experiment in Fig. 1 employed a mixture of normal troponin and CBMII-Tn. An extrapolation of these results suggests that for a thin filament with normal troponin only, the MgATPase rate will not increase in proportion to Ca2+ binding as the free Ca2+concentration is increased. Fig. 3 A shows the normalized MgATPase rate as a function of the free Ca2+concentration in the presence of either low myosin S1 concentrations as were also present in Fig. 1 (Fig. 3, □) or in the presence of much higher myosin S1 concentrations (Fig. 3,×). The rightmost two curves show the difference between Ca2+ binding (short dashes) and adjacent Ca2+ pair binding (solid curve, fit to MgATPase data (□)) according to Fig.1 under conditions where myosin·myosin cooperativity is precluded by low myosin S1 concentrations. The K app from the ATPase curve underestimates the true binding constant, but this discrepancy is small, 3.7 versus 2.4 × 105m−1 for K binding versus K app. The relationship between these curves is determined by Fig. 1; if the short dash curve in Fig. 3 A is set as the independent variable and the solid curve as the dependent variable, then a graph describing the data in Fig. 1 is the result. However, the lines actually were obtained by a best fit of Equation 1 to the experimental data (□). Assuming the MgATPase rate is proportional to the fraction of adjacent troponin pairs with Ca2+ on both sides, then the best fit regulatory site Ca2+ affinity is 3.7 ± 0.6 × 105m−1 and the cooperativity parameter Y = 3.4 ± 0.9. The mean value for Y from 10 such experiments was 5 ± 1, corresponding to a Hill coefficient of 2.2 (3Tobacman L.S. Sawyer D. J. Biol. Chem. 1990; 265: 931-939Abstract Full Text PDF PubMed Google Scholar) and similar to the level of cooperativity reported previously (1Tobacman L.S. Biochemistry. 1987; 26: 492-497Crossref PubMed Scopus (15) Google Scholar, 3Tobacman L.S. Sawyer D. J. Biol. Chem. 1990; 265: 931-939Abstract Full Text PDF PubMed Google Scholar, 13Tobacman L.S. Lee R. J. Biol. Chem. 1987; 262: 4059-4064Abstract Full Text PDF PubMed Google Scholar, 26Tobacman L.S. J. Biol. Chem. 1988; 263: 2668-2672Abstract Full Text PDF PubMed Google Scholar, 27Lin D. Bobkova A. Homsher E. Tobacman L.S. J. Clin. Invest. 1996; 97: 2842-2848Crossref PubMed Scopus (103) Google Scholar). The analysis in Fig. 3 A indicates that there is little difference in the cooperative shapes for Ca2+ binding and for Ca2+ pair binding (the solid and short dash curves are equally steep). Similarly, if Y is set at a noncooperative value of 1, both curves are less steep but they remain parallel, and the relationship between them is still consistent with Fig. 1 (not shown). This indicates that Fig. 1 is consistent with the data in Fig. 3 A, but only if overall Ca2+binding to the thin filament regulatory sites is cooperative. Since this process is known to have little cooperativity for reconstituted thin filaments (3Tobacman L.S. Sawyer D. J. Biol. Chem. 1990; 265: 931-939Abstract Full Text PDF PubMed Google Scholar, 4Huynh Q. Butters C.A. Leiden J.M. Tobacman L.S. Biophys. J. 1996; 70: 1447-1455Abstract Full Text PDF PubMed Scopus (23) Google Scholar, 28Rosenfeld S.S. Taylor E.W. J. Biol. Chem. 1985; 260: 252-261Abstract Full Text PDF PubMed Google Scholar, 29Zot H.G. Potter J.D. J. Muscle Res. Cell Motil. 1987; 8: 428-436Crossref PubMed Scopus (34) Google Scholar), some other explanation will be needed to rationalize the larger cooperativity observed for MgATPase activationversus the free Ca2+ concentration (Fig.3 A and Ref. 1Tobacman L.S. Biochemistry. 1987; 26: 492-497Crossref PubMed Scopus (15) Google Scholar). Another source of cooperativity in MgATPase assays is effects of myosin S1 on the thin filament. Careful studies of Weber and co-workers (30Bremel R.D. Murray J.M. Weber A. Cold Spring Harbor Symp. Quant. Biol. 1972; 37: 267-275Crossref Google Scholar) using skeletal muscle regulatory proteins have shown increased MgATPase rates, increased Ca2+ affinity, and apparent Ca2+ affinity (32Murray J.M. Weber A. Mol. Cell. Biochem. 1980; 35: 11-15Crossref Scopus (20) Google Scholar, 33Murray J.M. Weber A. Bremel R.D. Carafoli E. Calcium Transport in Contraction and Secretion. Elsevier Science Publishers B.V., Amsterdam1975: 489-496Google Scholar). These effects are observed when the myosin concentration is high relative to actin or when conditions favor strong actin–myosin bond formation (34Murray J.M. Knox M.K. Trueblood C.E. Weber A. Biochemistry. 1982; 21: 906-915Crossref PubMed Scopus (37) Google Scholar). Fig. 3 B shows the potentiating effect of high myosin S1 concentrations on the thin filament-myosin S1 MgATPase rate using cardiac regulatory proteins. For an actin concentration of 5 μm, the MgATPase rate deviated from linearity when the myosin S1 concentration was >3 μm. This deviation correlated with a shift in the Ca2+ K app in MgATPase versus pCa experiments. There was no shift for 3 μm myosin S1 (not shown), a small shift for 5 μm myosin S1 (not shown), and a 2.5-fold shift in K app in the presence of 10 μm myosin S1 (Fig. 3 A, ×). The apparent Ca2+ affinity from these and other titrations was 2.4 ± 0.2 × 105m−1 in the presence of 0.3 μm myosin S1 and 6.0 ± 0.9 × 105m−1 in the presence of 10 μm myosin S1. Comparison among the three curves in Fig. 3 Ashows that MgATPase rate activation of 10 μm myosin S1 (×) occurs at even lower Ca2+ concentrations than the calculated Ca2+ saturation of the regulatory sites (short dashes) when the myosin concentration is low. If the high myosin S1 (×) versus low myosin S1 (□) MgATPase rate shift had occurred without any change in true Ca2+ binding, MgATPase activation would precede fractional Ca2+ binding. This would be the opposite of the relationship in Fig. 1, a convex rather than a concave curve. This possibility is evaluated and excluded by Fig. 3 C, which shows fractional MgATPase activation as a function of bound Ca2+. Even in the presence of high myosin S1 concentrations that "potentiate" the thin filament, Ca2+ binding precedes fractional activation. Therefore, the shift seen in Fig. 3 A (× versus □) involves a myosin-induced increase in Ca2+ affinity. However, there may also be some change in the precise relationship between fractional Ca2+ binding and fractional MgATPase rate activation; Fig.3 C appears to show less deviation from linearity than does Fig. 1. In this regard, it should be noted that strongly bound cross-bridges can activate the thin filament under appropriate conditions, even in the absence of any Ca2+ binding (31Bremel R.D. Weber A. Nat. New Biol. 1972; 238: 97-101Crossref PubMed Scopus (494) Google Scholar). When only 25% of the troponins on the thin filament are capable of binding Ca2+, i.e. 75% of the troponin is of the form CBMII-Tn, a gradual increase in the Ca2+ concentration produces a small level of activation that is shown in Fig.4. The figure is a normalized composite of four experiments, and in all of them the noise precluded any assessment ofY. The data is noisy because a 25:75 ratio of troponin:CBMII-Tn produces only a low MgATPase rate (Fig. 1); the average Ca2+-saturated rate is twice the EGTA rate for these data sets. The solid curve is a noncooperative binding isotherm. Comparison of the data points to this theoretical curve suggests that cooperativity may actually be present (the data deviates from the curve), but this may be an artifact of the normalization of each data set. The K app could be measured with enough precision, 4.3 ± 1.2 × 105m−1, to permit comparison to the value found for thin filaments with fully normal troponin, 2.4 ± 0.2 × 105m−1 (n = 10, with representative data shown in Fig. 3 A, □). This was unexpected, since the CBMII-Tn might have cooperatively interacted with adjacent troponin molecules to decrease Ca2+ affinity. It is unclear why a modest increase in apparent affinity occurred instead, but the effect is small in any case. The thin filament has at least three conformations: an inhibited state in the presence of EGTA, a Ca2+-induced state, and an active state observed in the presence of strongly binding myosin cross-bridges (35Lehman W. Craig R. Vibert P. Nature. 1994; 368: 65-67Crossref PubMed Scopus (271) Google Scholar, 36Vibert P. Craig R. Lehman W. J. Mol. Biol. 1997; 266: 8-14Crossref PubMed Scopus (381) Google Scholar, 37Holmes K.C. Biophys. J. 1995; 68: 2s-7sPubMed Google Scholar). These structures have been compared with three-dimensional reconstructions of myosin S1-decorated thin filaments (38Milligan R.A. Whittaker M. Safer D. Nature. 1990; 348: 217-221Crossref PubMed Scopus (321) Google Scholar, 39Rayment I. Holden H.M. Whittaker M. Yohn C.B. Lorenz M. Hol
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