Abstract
Crayfish tail muscle troponin C (TnC) has been fractionated into its five components and the Ca2+-binding properties of the two major isoforms (alpha and gamma) determined by equilibrium dialysis. alpha-TnC contains one Ca2+-binding site with a binding constant of 1 x 10(6) M-1 and one Ca2+ site with a binding constant of 1 x 10(4) M-1. In the complex of alpha-TnC with troponin I (TnI) or with TnI and troponin T (TnT), both sites bind Ca2+ with a single affinity constant of 2-4 x 10(6) M-1. gamma-TnC contains two Ca2+-binding sites with a binding constant of 2 x 10(4) M-1. In the gamma-TnC.TnI and gamma-TnC.TnI.TnT complexes, the binding constant of one of the sites is increased to 4-5 x 10(6) M-1, while Ca2+ binding to the second site is hardly affected (KCa = 4-7 x 10(4) M-1). In the presence of 10 mM MgCl2, the two Ca2+-binding sites of both TnC isoforms exhibit a 2-3-fold lower affinity. Assuming competition between Ca2+ and Mg2+ for these sites, their binding constants for Mg2+ were 120-230 M-1. In the absence of Ca2+, however, alpha-TnC and gamma-TnC bind 4-5 mol of Mg2+/mol with a binding constant of 1 x 10(3) M-1. These results suggest that the effect of Mg2+ on Ca2+ binding at the two Ca2+ sites is noncompetitive, i.e. Mg2+ does not bind directly to these sites (Ca2+-specific sites). Since the formation of the complex of crayfish TnI with alpha-TnC or gamma-TnC increases significantly the affinity of one of their two Ca2+-specific sites, I conclude that the binding of Ca2+ to only one site (regulatory Ca2+-specific site) controls the Ca2+-dependent interaction between crayfish TnCs and TnI.
Highlights
Crayfish tailmuscle troponin C (TnC) habseen frac- in TnC are transmitted to the other subunits of Tn, to Tm, tionated intoits five components and theCa2+-binding and to actin, leading to the activation of myosin ATPase and properties of the two major isoforms deter- tension development
The results presented in miniprintat the endof this paper demonstrate a pronounced polymorphism in crayfish TnC
The five isoforms of this protein differ in their net charge as observed by electrophoresis in the absence of SDS (Fig. 4, insets), and in their relative hydrophobicity as seen during reversed-phase HPLC (Fig. 5)
Summary
0 1989 by The American Society for Biochemistry and Molecular Biology, Inc. Vol 264, No 30, Issue of October 25, pp. 18240-18246,1989 Printed in U.S A. 0 1989 by The American Society for Biochemistry and Molecular Biology, Inc. Vol 264, No 30, Issue of October 25, pp. Resolution and Calcium-bindingProperties of the Two Major Isoforms of TroponinC from Crayfish*. (Received for publication, January 25, 1989, and in revised form, July 10, 1989)
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