Use of stable analogs of myosin ATPase intermediates for kinetic studies of the "weak" binding of myosin heads to F-actin

It is known that ternary complexes of myosin subfragment 1 (S1) with ADP an d the P-i analogs beryllium fluoride (BeFx) and aluminum fluoride (AlF4-) a re stable analogs of the myosin ATPase intermediates M*.ATP and M**.ADP.P-i , respectively. Using kinetic approaches, we compared the rate of formation of the complexes S1.ADP.BeFx and S1.ADP.AlF4-, in the absence and in the p resence of F-actin, as well as of the interaction of these complexes with F -actin. We show that in the absence of F-actin the formation of S1.ADP.BeFx occurs much faster (3-4 min) than that of S1.ADP.AlF4-; (hours). The forma tion of these complexes in the presence of F-actin led to dissociation of S i from F-actin, this process being monitored by a decrease in light scatter ing. The light scattering decrease of the acto-S1 complex occurred much fas ter after addition of BeFx (during 1 min) than after addition of AlF4- (mor e than 20 min). In both cases the light scattering of the acto S1 complex d ecreased by 40-50%, but it remained much higher than: that of F-actin measu red in the absence of S1:, The interaction of the S1.ADP.BeFx and S1.ADP.Al F4-; complexes with F-actin was studied by the stopped-flow technique with high time resolution (no more than 0.6 sec after mixing of S1 with F-actin) . We found that the binding of S1.ADP.BeFx or S1.ADP.AlF4-; to F-actin is a ccompanied by a fast increase in light scattering, but it does not affect t he fluorescence of a pyrene label specifically attached to F-actin. We conc lude from these data that within this time range a "weak" binding of the S1 .ADP.BeFx and S1.ADP.AlF4-; complexes to F-actin occurs without the subsequ ent transition of the "weak" binding state to the "strong" binding state. C omparison of the light scattering kinetic curves shows that S1.ADP.AlF4-, b inds to F-actin faster than S1.ADP.BeFx does: the second-order rate constan ts for the "weak" binding to F-actin are (62.8 +/- 1.8) 10(6) M-1.sec(-1) i n the case of S1.ADP.AlF4- and (22.6 +/- 0.4).10(6) M-1 sec(-1) in the case of S1.ADP.BeFx. We conclude that the stable ternary complexes S1.ADP.BeFx and S1.ADP.AlF4-; can be successfully used for kinetic studies-of the "weak " binding of the myosin heads to F-actin.