CLONING AND EXPRESSION OF SALMON CARDIAC TROPONIN-C - TITRATION OF THE LOW-AFFINITY CA2-BINDING SITE USING A TRYPTOPHAN MUTANT()

Activation of cardiac actomyosin ATPase requires the occupation of the single low-affinity Ca2+-binding site of troponin C (cTnC). Previousl y, we demonstrated pronounced differences between mammals and cold-wat er salmonid fish in the Ca2+ sensitivity of cardiac preparations, part icularly in relation to temperature [Churcotte, C., Moyes, C. D., Bald win, K., Bressler, B., & Tibbits, G. F. (1994) Am. J. Physiol. 267, R6 2-R70]. In this study, we examine the extent to which cTnC structure c ould account for the observed differences in myofibrillar Ca2+ sensiti vity. Salmonid (Oncorhynchus mykiss) cTnC was cloned, sequenced, and e xpressed in Escherichia coli as a maltose-binding protein fusion. The coding region has 87% homology with human cTnC cDNA and differs in 13 of 161 amino acid residues from the human/bovine/porcine isoform. The sequence corresponding to the single regulatory Ca2+-binding site II i s completely homologous to that of mammals. The protein expressed exhi bits optical properties similar (circular dichroism, intrinsic fluores cence) to those of cTnC purified from salmonid (Salmo salar) and bovin e ventricle. A single tryptophan residue was introduced into the inact ive Ca2+-binding site I (ScTnC-FW27) to facilitate Ca2+ titration. The Ca2+-binding constant (K-1/2 = 5.33 pCa units) was within the range r eported for the low-affinity sites of mammalian cTnC. Although differe nces in TnC primary structure are striking, Ca2+ affinity of intact ca rdiac myofibrils is likely influenced by interactions with other tropo nin proteins.