Investigation of the effects of phosphorylation of rabbit striated muscle alpha alpha-tropomyosin and rabbit skeletal muscle troponin-T.

FPLC has been employed to prepare the phosphorylated and unphosphorylated forms of rabbit striated muscle alpha alpha-tropomyosin (TM), and the major isoform of rabbit fast-skeletal-muscle troponin-T (Tn-T2f) and corresponding chymotryptic fragment T1 (residues 1-158), in order to investigate the effects which these in vivo modifications have on thin filament function. In all instances, no significance could be attributed to the presence of a phosphate moiety on acetyl serine 1 of Tn-T (or fragment T1). As expected, fragment T1 increased the relative viscosities of solutions of unphosphorylated alpha alpha-TM, but this induction was noticeably lower for phosphorylated alpha alpha-TM. In affinity chromatography experiments, fragment T1 bound equally well to either form of alpha alpha-TM, but the interaction between fragment T2 (residues 159-259) and phosphorylated alpha alpha-TM was strengthened relative to the control. In the presence of alpha alpha-TM (unphosphorylated), fragment T1 was found to down regulate the actin-activated myosin-S1 MgATPase activity, indicating that this portion of Tn-T possesses modulatory properties. Under the same conditions, less inhibition was observed with phosphorylated alpha alpha-TM. When the two different forms of alpha alpha-TM were reconstituted into a complete regulatory system, the activation of myosin-S1 was double for those thin filaments containing the phosphorylated molecule. Dephosphorylation of the phospho alpha alpha-TM reduced the rates to control values. In ATPase Ca2+ titrations, these systems exhibited no difference in the co-operativity of activation and little or no difference in the pCa2+ 1/2 value. Developmentally linked changes in the steady-state phosphorylation of alpha alpha-TM could be a mechanism to increase the activating propensity of thin filaments, by modifying the functional properties of the T1 section of Tn-T.