DETERMINATION OF THE CA2-C FROM EEL SKELETAL-MUSCLE AND POSITIONING OF THE SINGLE TRYPTOPHAN IN THE PRIMARY STRUCTURE( AND MG2+ AFFINITY CONSTANTS OF TROPONIN)

The complete amino acid sequence of troponin C (ETnC) from the white m uscle of the European eel has been determined by Edman degradation pro cedures. Its single tryptophan residue is situated in helix H at amino acid position 152 of the aligned sequence; the tryptophan is the firs t residue on the C-terminal side of Ca2+ binding loop IV. The increase of tryptophan fluorescence emission intensity occurring upon titratio n 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 Ca2+-Mg2+ sites of the sec ond domain of fast skeletal muscle TnCs. Moreover, a direct competitio n 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 affinit y constants of the Ca2+ binding sites of the regulatory domain, ETnC w as labelled with dansylaziridine (Danz). The Danz fluorescent signal w as used to estimate the affinity constants of ETnC-Danz for Ca2+ and a lso for Mg2+ (assuming a competitive behaviour between these two metal ions). The calculated affinity constants are K-(Ca)=9.4 10(5) M(-1) a nd K-(Mg)=2.0 10(2) M(-1), respectively. These values are typical of t he Ca2+-specific sites of the regulatory domain of fast skeletal muscl e TnCs.