Calcium binding and fluorescence measurements of dansylaziridine-labelled troponin C in reconstituted thin filaments

Henry G. Zot, James D. Potter

Research output: Contribution to journalArticle

32 Scopus citations

Abstract

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.

Original languageEnglish (US)
Pages (from-to)428-436
Number of pages9
JournalJournal of Muscle Research and Cell Motility
Volume8
Issue number5
DOIs
StatePublished - Oct 1987

ASJC Scopus subject areas

  • Biochemistry
  • Physiology
  • Cell Biology

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