KINETICS OF THE INTERACTION OF 2'(3')-O-(N-METHYLANTHRANILOYL)-ATP WITH MYOSIN SUBFRAGMENT-1 AND ACTOMYOSIN SUBFRAGMENT-1 - CHARACTERIZATION OF 2 ACTO.S1.ADP COMPLEXES

We have used a fluorescent analogue of ATP, mantATP [2'(3')-O-(N-methylanthraniloyl)-adenosine 5'-triphosphate; Hiratsuka, T. (1983) Biochim. Biophys. Acta 742, 496-508], and made a detailed kinetic study of the interaction of mantATP and mantADP with S1 and acto.S1. We have shown that these analogues behave like ATP and ADP, respectively. In addition, we have demonstrated that this analogue can distinguish between two acto.S1 complexes, the A-M.N (attached) and A.M.N (rigor-like) states [Geeves, M. A., Goody, R. S., & Gutfreund, H. (1984) J. Muscle Res. Cell Motil. 5, 351-361]. Previously, these two states were observed with a pyrene label on Cys 374 of actin. This isomerization can now be monitored at two spatially distinct sites on the ternary complex, indicative of a major conformational change in the ternary complex. Also, we have measured the rate of ADP dissociation from both A-M.N and A.M.N directly and shown these to differ by a factor of 1000. Thus the results presented here support the model of Geeves et al. and are consistent with the A-M.N to A.M.N transition being coupled to the force-generating event of the crossbridge cycle.