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Modulation of Troponin‐C Binding to Troponin‐T by Ca 2+ , Probed by Fluorescence
Author(s) -
Lin TsungI,
Mayadevi M.,
Dowben R. M.
Publication year - 1993
Publication title -
journal of the chinese chemical society
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.329
H-Index - 45
eISSN - 2192-6549
pISSN - 0009-4536
DOI - 10.1002/jccs.199300098
Subject(s) - chemistry , troponin c , fluorescence , titration , binding constant , troponin t , coumarin , biophysics , troponin , binding site , analytical chemistry (journal) , stereochemistry , nuclear chemistry , chromatography , crystallography , biochemistry , inorganic chemistry , organic chemistry , psychology , physics , quantum mechanics , psychiatry , myocardial infarction , biology
The effects of Ca 2+ on the binding interaction between troponin‐C (TnC) and troponin‐T (TnT) and between TnC and troponin‐I (TnI) were studied in both the binary complexes and the presence of other subunits and tropomyosin (Tm). Rabbit skeletal TnC was labeled with 7‐dimemyl‐amino‐4‐methyl‐3‐ N′ ‐meleimido‐coumarin (DACM). The fluorescence titration curve of the labeled TnC with Ca 2+ showed a biphasic response; fluorescence was quenched (by 31%) initially but increased (by 64%) in the second phase. In the presence of CaCl 2 (3 mM), titrating labeled TnC with TnT caused further fluorescence enhancement (32–34%) of DACM by TnT. In the absence of Ca 2+ , TnT bound weakly to TnC (K a < 10 3 M −1 ) whereas in the presence of Ca 2+ , the TnT‐TnC binding affinity increased almost 1000 fold (K a = 0.42 × 10 6 M −1 ). The affinity constant of unlabeled TnC for TnT is 10 – 15 times that of the labeled complex (K a = (3.4∼ 5.1) × 10 6 M −1 ); this value was determined from the binding isotherm of a mixed system consisting of labeled and unlabeled TnC at a fixed ratio upon titration with TnT. Adding Tm to the system weakened the binding of TnT to TnC by at least 50 – 60 percent. Adding Tnl to DACM‐TnC alone also enhanced the fluorescence (20–22%). An affinity constant 10 8 . M −1 for TnC‐Tnl binary complex was obtained. If Tn‐I and the labeled TnC were premixed first in 1:1 stoichiometry, then titrated with TnT, a further fluorescence increase (34%) similar to that in the absence of TnI was observed. The fit of the binding curve shows that K a of TnT to TnC increased (1.5 × 10 6 M −1 ). When TnI was added to the TnC‐TnT complex at the end of titration when the fluorescence binding curve became leveled, quenching (12%) occurred. The latter result indicates that TnI competes with TnT for the same binding sites on TnC. As binding of TnC is at least 100 times as strong to TnI as to TnT, a quenching effect is observed. Furthermore, the conformation of TnC in the TnC‐TnI binary complex may vary from that of TnC alone; binding of TnT to TnC is greatly enhanced (directly or in directly by TnI) in the TnC‐TnI complex. These results indicate that the variation of binding affinity between TnC and TnT as modulated by Ca 2+ may play an important role in the Ca +2 ‐regulation mechanism of muscle contraction.