Interrelation among the three components of troponin and tropomyosin studied by their kinetic effects on the ATPase reaction of actomyosin.

Abstract

The effects of tropomyosin and the three components of troponin on the Mg2+-activated ATPase [EC 3.6.1. 3] activity of reconstituted actomyosin were studied over a wide range of MgATP concentrations (0.002–1 mM) in 40 mM KCI at pH 7.2 and 25°C. The following results were obtained. 1. In the absence of tropomyosin, each of the two components of troponin with molecular weights of 23, 000 (TN-I) and 37, 000 (TN-P) activated the ATPase activity of actomyosin at MgATP concentrations above 0.1 mM and inhibited it below 0.1 mM. A mixture of both the components activated the ATPase activity considerably at MgATP concentrations above 0.04–0.05 mM and inhibited it slightly below 0.04 mM. These effects on ATPase were unaffected by Ca2+. The component of troponin with a molecular weight of 19, 000 (TN-C) neutralized both the activating and inhibitory activities of TN-I, TN-P or a mixture of these two components, regardless of the Ca2+ concentration, although TN-C alone had no effect on the ATPase activity of actomyosin. 2. Tropomyosin activated the ATPase activity of actomyosin at low concentrations of MgATP, and inhibited it at high concentrations of MgATP both in the presence and absence of Ca2+. Thus, tropomyosin greatly enhanced the substrate-inhibition of the ATPase of actomyosin. 3. In the presence of tropomyosin, TN-I inhibited the ATPase activity irrespective of the Ca2+ concentration, so that it reduced the concentration at which MgATP became inhibitory for the ATPase, to the level obtained with the complete relaxing protein system in the absence of Ca2+, while TN-C had no effect on the ATPase activity even in the presence of tropomyosin. In the presence of tropomyosin, TN-P slightly inhibited the ATPase activity at low concentrations of MgATP but activated it at high concentrations of MgATP. 4. When TN-C was added to the TN-I-tropomyosin system, the Ca2+-sensitivity of the ATPase of actomyosin was only incompletely restored, since the inhibitory effect of the TN-C-TN-I-tropomyosin system in the absence of Ca2+ was weak, and since the ATPase was not activated in the presence of Ca2+ and at high concentrations of MgATP. The inhibitory activity of the TN-I-tropomyosin system was unaffected by addition of TN-P. When the TN-P-TN-C-tropomyosin system was added to actomyosin, Ca2+-sensitivity of the ATPase was also observed, but the ATPase activity at high MgATP concentrations was higher in the absence of Ca2+ than in its presence. 5. The relaxing protein activities were completely reproduced, only when tropomyosin and all the three components of troponin were added to actomyosin.