Determination of the Ca2+ and Mg2+ affinity constants of troponin C from eel skeletal muscle and positioning of the single tryptophan in the primary structure

Jean Marie François, Charles Gerday, Franklyn G. Prendergast, James D. Potter

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9 Scopus citations

Abstract

The complete amino acid sequence of troponin C (ETnC) from the white muscle of the European eel has been determined by Edman degradation procedures. Its single tryptophan residue is situated in helix H at amino acid position 152 of the aligned sequence; the tryptophan is the first residue on the C-terminal side of Ca2+ binding loop IV. The increase of tryptophan fluorescence emission intensity occurring upon titration of ETnC with Ca2+ has been used to determine the affinity constants of ETnC for Ca2+. The calculated affinity of ETnC for Ca2+ results in a K(Ca) of 1.3 107 M-1, typical of the Ca2+-Mg2+ sites of the second domain of fast skeletal muscle TnCs. Moreover, a direct competition between Ca2+ and Mg2+ was also observed. The calculated affinity of ETnC for Mg2+ is K(Mg)=1.2 103 M-1. In order to probe the affinity constants of the Ca2+ binding sites of the regulatory domain, ETnC was labelled with dansylaziridine (Danz). The Danz fluorescent signal was used to estimate the affinity constants of ETnC-Danz for Ca2+ and also for Mg2+ (assuming a competitive behaviour between these two metal ions). The calculated affinity constants are K(Ca)=9.4 105 M-1 and K(Mg)=2.0 102 M-1, respectively. These values are typical of the Ca2+-specific sites of the regulatory domain of fast skeletal muscle TnCs.

Original languageEnglish
Pages (from-to)585-593
Number of pages9
JournalJournal of Muscle Research and Cell Motility
Volume14
Issue number6
DOIs
StatePublished - Dec 1 1993
Externally publishedYes

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ASJC Scopus subject areas

  • Physiology
  • Endocrinology
  • Clinical Biochemistry
  • Cell Biology

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