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.
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 10(7) M-1, typical of the Ca(2+)-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 10(3) 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 10(5) M-1 and K(Mg) = 2.0 10(2) M-1, respectively. These values are typical of the Ca(2+)-specific sites of the regulatory domain of fast skeletal muscle TnCs.
François
J M
JM
Laboratoire de Biochimie, Université de Liège, Sart Tilman, Belgium.
Gerday
C
C
Prendergast
F G
FG
Potter
J D
JD
D000595:Amino Acid Sequence; D000818:Animals; D001388:Aziridines; D002118:Calcium / Q000378:metabolism*; D003619:Dansyl Compounds; D004524:Eels / Q000378:metabolism*; D005453:Fluorescence; D008274:Magnesium / Q000378:metabolism*; D008969:Molecular Sequence Data; D009132:Muscles / Q000737:chemistry* / Q000378:metabolism*; D011817:Rabbits; D017386:Sequence Homology, Amino Acid; D014336:Troponin / Q000032:analysis* / Q000737:chemistry / Q000378:metabolism*; D019209:Troponin C; D014364:Tryptophan / Q000032:analysis*
D001388:Aziridines; D003619:Dansyl Compounds; D014336:Troponin; D019209:Troponin C; C017173:dansylaziridine; D014364:Tryptophan; D008274:Magnesium; D002118:Calcium