The direct binding of Ca2+ to reconstituted thin filaments containing troponin C and the 5-dimethylaminonaphthalene-1-sulphonylaziridine (DANZ) fluorescent analogue of troponin C (TnCDANZ) was measured (25° C) at three Mg2+ concentrations. Biphasic Scatchard plots were found for all binding curves reflecting the binding of Ca2+ to high- and low-affinity sites of troponin. The binding of Ca2+ to the high-affinity sites had a greater sensitivity to Mg2+ (KMg=1×104m-1) than the low-affinity sites (KMg=1.2×103m-1). The fluorescence change of thin filaments reconstituted with TnCDANZ was titrated with Ca2+ in the same solutions used for binding assays. The Ca2+-dependent fluorescence change had nearly the same sensitivity to Mg2+ (KMg=9.4×102m-1) as did Ca2+ binding to the low-affinity sites. The Ca2+ concentration at the midpoint of the fluorescence change was about 0.3 log units less than at the midpoint for Ca2+ binding to the low-affinity sites. A similar relationship between the fluorescence change and Ca2+ binding to the low-affinity sites of isolated TnCDANZ was measured (4° C). From these results the binding of Ca2+ to either low-affinity site is concluded to produce the fluorescence change. In comparison with the low-affinity sites of isolated troponin and troponin-tropomyosin complex, the low-affinity sites of reconstituted thin filaments were consistently lower in Ca2+ affinity.
ASJC Scopus subject areas
- Cell Biology