Calcium binding and fluorescence measurements of dansylaziridine-labelled troponin C in reconstituted thin filaments.
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 degrees 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 x 10(4)M-1) than the low-affinity sites (KMg = 1.2 x 10(3)M-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 x 10(2)M-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 degrees 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.
Department of Pharmacology, University of Miami School of Medicine, Florida 33101.
H G
HG
Zot
J D
JD
Potter
D008841:Actin Cytoskeleton / Q000378:metabolism*; D000199:Actins / Q000378:metabolism; D000818:Animals; D002118:Calcium / Q000493:pharmacokinetics*; D003599:Cytoskeleton / Q000378:metabolism*; D003619:Dansyl Compounds / Q000378:metabolism*; D005456:Fluorescent Dyes; D008274:Magnesium / Q000493:pharmacokinetics; D011817:Rabbits; D013050:Spectrometry, Fluorescence; D014336:Troponin / Q000031:analogs & derivatives* / Q000378:metabolism*
D000199:Actins; D003619:Dansyl Compounds; D005456:Fluorescent Dyes; D014336:Troponin; C043338:dansylaziridine-troponin C; D008274:Magnesium; D002118:Calcium