Coupling of calcium to the interaction of troponin I with troponin C from cardiac muscle.
The interaction of troponin I (CTnI) with troponin C (CTnC) from bovine cardiac muscle was studied using CTnC modified at Cys35 and Cys84 with the fluorescent probe 2-[(4'-iodoacetamido)-anilino]naphthalene-6-sulfonic acid (CTnCIAANS). The association constant for complex formation between the two proteins was determined at 20 degrees C in 0.4 M KCl, 1 mM DTT, 1 mM EGTA, and 25 mM MOPS, pH 7.2. In the presence of EGTA, Mg2+, and Ca2+ these constants were 1.46 x 10(7), 4.1 x 10(7), and 12.7 x 10(7) M-1, respectively, with corresponding free energy values of -9.62, -10.23, and -10.88 kcal mol-1. The CTnI-CTnCIAANS complex was stabilized by -0.61 kcal when the two Ca/Mg sites of CTnCIAANS were saturated with Mg2+ and by -1.26 kcal when all three Ca2+ sites were occupied by Ca2+. These results suggest that calcium activation in cardiac muscle may be accompanied by a coupling free energy of -0.65 kcal. This value is a factor of 4 smaller than the value previously determined, using a similar method, for the (troponin I).(troponin C) complex from skeletal muscle [Wang, C.-K., & Cheung, H.C. (1985) Biophys. J.48, 727-739]. Since CTnC has only one Ca(2+)-specific site and troponin C from skeletal muscle has two such sites, the present result is a factor of 2 smaller than that for the skeletal complex on the basis of a single specific site. Phosphorylation of CTnI by 3',5'-cyclic AMP-dependent protein kinase resulted in a decrease of the association constants by a factor of 2.5-3.5.(ABSTRACT TRUNCATED AT 250 WORDS)
Liao
R
R
Graduate Program in Biophysical Sciences, University of Alabama at Birmingham 35294-2041.
Wang
C K
CK
Cheung
H C
HC
D000818:Animals; D002118:Calcium / Q000378:metabolism*; D002417:Cattle; D005456:Fluorescent Dyes; D066298:In Vitro Techniques; D008956:Models, Chemical; D009206:Myocardium / Q000378:metabolism*; D010766:Phosphorylation; D011485:Protein Binding; D013050:Spectrometry, Fluorescence; D013816:Thermodynamics; D014336:Troponin / Q000378:metabolism*; D019209:Troponin C; D019210:Troponin I
D005456:Fluorescent Dyes; D014336:Troponin; D019209:Troponin C; D019210:Troponin I; D002118:Calcium