Evidence for structural changes in vertebrate thick filaments induced by calcium

Abstract

Paired sedimentation studies of isolated, native thick filaments at pH 6.8, I = 0.12 and in the presence of 0.3 m m-free Mg 2+ show that the sedimentation coefficient increases with Ca 2+ concentration (pCa † † Abbreviations used: pCa, negative logarithm of molar concentration of free Ca 2+; DTNB light chain, the light chain removed from myosin by treatment with 5,5′-dithiobis (2-nitrobenzoic acid; HMM, heavy meromyosin; HMMS-1, heavy meromyosin subfragment-1. midpoint = 5.5), leveling off at pCa 4.7. The addition of ATP or ADP (5 m m) has no effect on the hydrodynamic changes induced by Ca 2+. At much higher free Mg 2+ concentrations (5 m m), the midpoint of the transition is shifted to pCa = 5.3. Viscosity measurements of the filament system under comparable conditions reveal a decrease in the relative viscosity over the same range of Ca 2+ concentration. Synthetic filaments prepared from purified myosin free of C-protein also show the same behavior. Native filaments from which myosin heads have been removed by treatment with papain do not show Ca 2+ dependence. The dependence of the sedimentation coefficient of filament on protein concentration, as measured by differential sedimentation, is unaffected by Ca 2+, indicating that the changes in hydrodynamic properties are probably not related to aggregation of the filaments. The Ca 2+ effects are reversible and are not observed on replacing Ca 2+ by Mg 2+. Binding studies carried out at low ionic strength reveal two binding sites for Ca 2+ ( K a = 1.7 × 10 5 m −1) per mole myosin within the filament and evidence is presented showing that the DTNB light chain is the site of binding. The combined results are interpreted as indicating that thick filaments of vertebrate muscle undergo conformational changes at physiological levels of Ca 2+ and provide evidence for a Ca 2+-sensitive regulatory mechanism at the level of the thick filament.