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.
Heeley
D H
DH
Department of Biochemistry, Memorial University, St John's, Newfoundland, Canada.
D000199:Actins / Q000494:pharmacology; D000818:Animals; D017301:Ca(2+) Mg(2+)-ATPase / Q000378:metabolism; D002846:Chromatography, Affinity; D002851:Chromatography, High Pressure Liquid; D002918:Chymotrypsin / Q000378:metabolism; D009132:Muscles / Q000737:chemistry*; D015879:Myosin Subfragments / Q000378:metabolism; D009218:Myosins / Q000378:metabolism; D010446:Peptide Fragments / Q000302:isolation & purification / Q000378:metabolism / Q000494:pharmacology; D010766:Phosphorylation; D011817:Rabbits; D014335:Tropomyosin / Q000378:metabolism*; D014336:Troponin / Q000378:metabolism* / Q000494:pharmacology; D020107:Troponin T; D014783:Viscosity
D000199:Actins; D015879:Myosin Subfragments; D010446:Peptide Fragments; D014335:Tropomyosin; D014336:Troponin; D020107:Troponin T; D002918:Chymotrypsin; D017301:Ca(2+) Mg(2+)-ATPase; D009218:Myosins