Investigation of the effects of phosphorylation of rabbit striated muscle αα‐tropomyosin and rabbit skeletal muscle troponin‐T

Abstract

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.