The effect of nucleotide on the binding of myosin subfragment 1 to regulated actin.

Previous studies have shown that in the presence of ADP, myosin subfragment 1 (S-1) binds with positive cooperativity to the troponin-tropomyosin-actin complex (regulated actin).This binding is much more cooperative in the absence than in the presence of ca'+.Based on these data, we proposed a model (Hill, T. L., Eisenberg, E., and Greene, L. E. (1980) Proc.Natl.Acad.Sci.U. S. A. 77, 7209-7213) in which the tropomyosinactin units in regulated actin exist in two states, a state which binds S-1 weakly and a state which binds S-1 strongly.This model predicts that the equilibrium constant between the weak and strong states of the tropomyosin-actin units is an intrinsic property of the regulated actin filament.Therefore, its value should not change when measured with different nucleotides bound to S-1.To test this prediction, the equilibrium constant was measured in the presence of S-1, S-1.ADP, and S-lOAMP-PNP, with and without Ca'+.The results show that, in all cases, S-1 binds with positive cooperativity to regulated actin.The observed cooperativity is consistent with the model since it was possible to fit the data obtained in the presence of different nucleotides with the same value for the equilibrium constant between the weak and strong states of the tropomyosin-actin units.However, the nucleotide bound to S-1 appears to affect the observed cooperativity by changing the affinity of S-1 for the strong and weak states.The troponin-tropomyosin complex strongly inhibits the binding of S-1'.ADP to actin in the absence of Ca", and to a lesser extent in the presence of Ca2+, when most of t h e S-1 binding sites on actin are unoccupied.In contrast, when most of these sites are occupied, troponin-tropomyosin causes a slight enhancement in S-1 binding.The binding of S-1 .ADP to regulated actin therefore shows positive cooperativity both in the presence and absence of Ca2+ (1).To account for these data we proposed a model (2) in which each tropomyosintroponin-actin unit along the F-actin filament (comprised of one troponin, one tropomyosin, and seven F-actin monomers) exists in two states, a weak binding state and a strong binding state, which differ in their ability to bind S-1.There are two sources of cooperativity in this model: first, the seven actin sites change state as a single unit (group cooperativity); second, there are attractive nearest neighbor interactions be-* The costs of publication of this article were defrayed in part by the payment of page charges.