Apparent Ca2+-binding constant (K-app) of Caenorhabditis elegans troponin C
(CeTnC) was determined by a fluorescence titration method. The K-app of th
e N-domain Ca2+-binding site of CeTnC was 7.9 +/- 1.6 x 10(5) M-1 and that
of the C-domain site was 1.2 +/- 0.6 x 10(6) M-1, respectively. Mg2+-depend
ence of the K-app showed that both Ca2+-binding sites did not bind competit
ively Mg2+. The Ca2+ dissociation rate constant (k(off)) of CeTnC was deter
mined by the fluorescence stopped-flow method. The k(off) of the N-domain C
a2+-binding site of CeTnC was 703 +/- 208 s(-1) and that of the C-domain si
te was 286 +/- 33 s(-1), respectively. From these values we could calculate
the Ca2+-binding rate constant (k(on)) as to be 5.6 +/- 2.8 x 10(8) M-1 s(
-1) for the N-domain site and 3.4 +/- 2.1 x 10(8) M (1) s(-1) for the C-dom
ain site, respectively. These results mean that all Ca2+-binding sites of C
eTnC are low affinity, fast dissociating and Ca2+-specific sites. Evolution
al function of TnC between vertebrate and invertebrate and biological funct
ions of wild type and mutant CeTnCs are discussed. (C) 2001 Elsevier Scienc
e B.V. All rights reserved.