Regulation of actomyosin ATPase by a single calcium-binding site on troponin C from crayfish.
Equilibrium-binding studies at 4 degrees C show that, in the instance of crayfish, troponin C contains only one Ca-binding site with an affinity in the range of physiological free [CA2+] (K = 2 X 10(5) M-1). At physiological levels of Mg2+, this site does not bind Mg2+. In the complexes of troponin C-troponin I, troponin and troponin-tropomyosin, the regulatory Ca-specific site exhibits a 10- to 20-fold higher affinity (K = 2-4 X 10(6) M-1). The latter affinity is reduced to that of troponin C upon incorporation of the troponin-tropomyosin complex into the actin filament (regulated actin), as determined at 4 degrees C by the double isotope technique. The Ca-binding constant is again shifted to a higher value (7 X 10(6) M-1) when regulated actin is associated with nucleotide-free myosin. Both crayfish myofibrils and rabbit actomyosin regulated by crayfish troponin-tropomyosin display a steep rise in ATPase activity with [Ca2+]. Comparison of the pCa/ATPase relationship and the Ca-binding properties at 25 degrees C for the crayfish troponin-regulated actomyosin indicates that while the threshold [Ca2+] for activation corresponds to the range of [Ca2+] where the regulatory site in its low affinity state (K = 1 X 10(5) M-1) starts to bind Ca2+ significantly, full activation is reached at [Ca2+] for which the Ca-specific site in its high affinity state (K = 3 X 10(6) M-1) approaches saturation. These results suggest that, in the actomyosin ATPase cycle, there are at least two calcium-activated states of regulated actin (one low and one high), the high affinity state being induced by interactions of myosin with actin in the cycle.
D000199:Actins / Q000378:metabolism; D000251:Adenosine Triphosphatases / Q000378:metabolism*; D000818:Animals; D003400:Astacoidea; D001665:Binding Sites; D002118:Calcium / Q000378:metabolism*; D000252:Calcium-Transporting ATPases / Q000378:metabolism; D007700:Kinetics; D009132:Muscles / Q000378:metabolism; D009210:Myofibrils / Q000378:metabolism; D009218:Myosins / Q000378:metabolism; D011485:Protein Binding; D011817:Rabbits; D014336:Troponin / Q000378:metabolism*; D019209:Troponin C
D000199:Actins; D014336:Troponin; D019209:Troponin C; D000251:Adenosine Triphosphatases; D009218:Myosins; D000252:Calcium-Transporting ATPases; D002118:Calcium