Studies on the regulatory complex of rabbit skeletal muscle: Contributions of troponin subunits and tropomyosin in the presence and absence of Mg2+ to the acto-S1 ATPase activity

Jennifer E. Van Eyk, Paul J. Cachia, Richard H. Ingraham, Robert S. Hodges

Research output: Contribution to journalArticlepeer-review

Abstract

The regulatory roles of the components of the troponin-tropomyosin complex in the presence and absence of Mg2+ on the acto-S1 ATPase have been examined. The effect of free Mg2+ on the inhibition of the acto-S1 ATPase by rabbit skeletal troponin (Tn) was studied at S1 to actin ratios ranging from 0.17:1 to 2.5:1. These studies were performed using two Mg2+ concentrations: 2.5 mM Mg2+-2.5 mM ATP, conditions considered to have low free Mg2+; and 5.0 mM Mg2+-2.5 mM ATP, conditions providing a high free Mg2+ concentration of ∼2.5 mM. In the presence of high free Mg2+ (2.5 mM ATP-5.0 mM MgCl2) the Tn inhibition of acto-S1-TM ATPase increased by approximately 40-50% over a range of S1 to actin ratios of 0.17:1 to 2.5:1. The effect of free Mg2+ on increasing quantities of Tn in the absence or presence of tropomyosin was studied independently at two S1 to actin ratios (1:1 and 2:1). In the absence of TM, at 5 mM Mg2+ there is an additional 38% (1:1 S1 to actin) or 37% (2:1) decrease in the ATPase activity by Tn compared to 2.5 mM Mg2+. Similarly, in the presence of TM and Tn, Mg2+ exerts its effect at both S1 to actin ratios. Significantly, the inhibition by the IT complex in the presence of TM is unaffected by free Mg2+. Furthermore, ultracentrifugation binding studies using14C-iodoacetamide-labeled Tn and TM established that the Tn-TM regulatory complex was firmly bound to F-actin at both Mg2+ concentrations, indicating that faciliation of binding to F-actin by Mg2+ is not responsible for the increased inhibition. Hence, it is concluded from the data that Mg2+ binding and by analogy Ca2+ binding to the Ca2+-Mg2+ sites of TnC promotes muscle relaxation by inducing inhibition of the actomyosin ATPase, whereas Ca2+ binding to the Ca2+-specific sites promotes contraction by potentiating the ATPase. The inhibition of the acto-S1-TM ATPase by TnT has also been further examined. The data indicate that TnT exerts the same level of inhibition upon the ATPase as TnI or Tn. The inhibitory activity requires TM, and occurs to the same extent under conditions where TM alone would have either a potentiating (2:1 S1 to actin) or an inhibitory (1:1 S1 to actin) effect upon the ATPase. In the presence of TM the IT complex is a more effective inhibitor than either TnI, TnT, or Tn. The inhibitory activity of the IT complex is partially released by TnC in the absence of Ca2+. These observations, in conjunction with those by Chong, Asselbergs, and Hodges, which showed that the inhibition by TnT is partially released by TnC plus Ca2+, indicate that the role of TnT involves more than anchoring Tn to the thin filament.

Original languageEnglish (US)
Pages (from-to)335-354
Number of pages20
JournalJournal of Protein Chemistry
Volume5
Issue number5
DOIs
StatePublished - Oct 1 1986

Keywords

  • Mg binding
  • acto-S1 ATPase
  • troponin T inhibition
  • troponin-tropomyosin

ASJC Scopus subject areas

  • Biochemistry

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