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Calcium-insensitive binding of heavy meromyosin to regulated actin at physiological ionic strength
Abstract:
Several conflicting reports have been made regarding the affinity of myosin heads (subfragment 1 and heavy meromyosin (HMM) for regulated actin (actin complexed with tropomyosin and troponin) at low ionic strength (mu = 18-50 mM) and whether or not this interaction is Ca2+ sensitive (Chalovich, J. M., and Eisenberg, E. (1982) J. Biol. Chem. 257, 2432-2437; Chalovich, J. M., and Eisenberg, E. (1984) Biophys. J. 45, 221a; Wagner, P. D., and Stone, D. B. (1983) Biochemistry 22, 1334-1342; and Wagner, P. D. (1984) Biochemistry 23, 5950-5956). Since the low ionic strengths used in the above studies do not represent the physiological ionic strength under which intact muscle exhibits Ca2+-dependent tension development, we investigated the possibility of whether a Ca2+-dependent regulated actin-HMM interaction could be observed at physiological ionic strength (mu = 134 mM, pH 7.4) and in the presence of ATP (at 23-24 degrees C). Direct binding of HMM to varied concentrations of regulated actin (87.7-221 microM free actin) was measured by sedimentation in an air-driven ultracentrifuge. Under the above conditions, we found that the regulated actin activation of HMM-Mg2+-ATPase was about 94% inhibited in the absence of Ca2+ although the association constant (Ka) is only moderately affected in the presence of Ca2+. These results are similar to those obtained by Chalovich and Eisenberg (1982 and 1984) with subfragment 1 and HMM, respectively, at low ionic strength and support their suggestion that in solution tropomyosin-troponin may not act totally by physically blocking the formation of cross-bridges with actin, but instead may act to inhibit a kinetic step in the overall ATPase rate. Whether this holds true in more intact systems (e.g. myosin, thick filaments) remains to be determined. Our results also show a good correlation between levels of ATPase activation and HMM binding by unregulated actin and in regulated actin in the presence of Ca2+.