Cardiac troponin C (C-TnC) was labeled with the sulfhydryl-specific fluorescent probe molecule 2-(4'-iodoacetamidoanilino)naphthalene-6-sulfonic acid at cysteine 35 and 84 to produce C-TnC(IA). This modified protein binds Ca2+, undergoes Ca2+-induced increases in α helix, and forms a complex with other troponin subunits as does unlabeled C-TnC. C-TnC(IA) undergoes a small fluorescence decrease with Ca2+ or Mg2+ binding to the two high affinity Ca2+, Mg2+ sites of C-TnC and a large biphasic ~ 2.1-fold fluorescence increase with Ca2+ binding to two lower affinity Ca2+-specific sites with K(Ca) of ~ 4.5 x 10:5 M-1 and ~ 5 x 102 M-1. C-TnC(IA) was formed in a complex with troponin I (TnI) and troponin T to form C-Tn(IA). This fluorescent reconstituted whole troponin undergoes a 25% decrease with Ca2+ binding to a Ca2+ -specific site of K(Ca) ~ 3 x 106 M-1. C-TnC, therefore, contains a single Ca2+-specific site of approximate equal affinity as the two Ca2+-specific regulatory sites of skeletal TnC. This Ca2+-specific site in C-TnC (like its two corresponding sites in S-TnC) undergoes an approximate 10-fold increase in affinity in whole troponin or when TnC is complexed with TnI. Since the two Ca2+-specific sites in skeletal troponin have been shown to be the regulatory sites of skeletal muscle contraction we suggest that this single Ca2+-specific site, of equal affinity, in C-TnC is the regulatory site of cardiac muscle contraction.
|Original language||English (US)|
|Number of pages||6|
|Journal||Journal of Biological Chemistry|
|State||Published - 1980|
ASJC Scopus subject areas
- Molecular Biology
- Cell Biology