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
✍ Scribed by Jean-Marie François; Charles Gerday; Franklyn G. Prendergast; James D. Potter
- Publisher
- Springer Netherlands
- Year
- 1993
- Tongue
- English
- Weight
- 862 KB
- Volume
- 14
- Category
- Article
- ISSN
- 0142-4319
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✦ Synopsis
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 Ca'+ has been used to determine the affinity constants of ETnC for Cal+. The calculated affinity of ETnC for Ca2+ results in a K,,,, of 1.3 lo7 Mm', typical of the Ca"-Mg'+ sites of the second domain of fast skeletal muscle TnCs. Moreover, a direct competition between Ca" and Mg"' was also observed. The calculated affinity of ETnC for Mg" is K,,,, = 1.2 10'Mml.
In order to probe the affinity constants of the Ca2+ binding sites of the regulatory domain, ETnC was labelled with dansylaziridine (Danz). The Dam fluorescent signal was used to estimate the affinity constants of ETnC-Danz for Cal' and also for Mg"
(assuming a competitive behaviour between these two metal ions). The calculated affinity constants are Ko,, = 9.4 lo5 Mm* and K,,,, = 2.0 lO"M-', respectively. These values are typical of the Ca"-specific sites of the regulatory domain of fast skeletal muscle TnCs.