Use of Stable Analogs of Myosin ATPase Intermediates for Kinetic Studies of the “Weak” Binding of Myosin Heads to F-Actin

E. V. Rostkova¹, L. N. Moiseeva¹, M. V. Teplova¹, O. P. Nikolaeva², and D. I. Levitsky^1,2*

¹Bach Institute of Biochemistry, Russian Academy of Sciences, Leninsky pr. 33, Moscow, 117071 Russia; fax: (095) 954-2732; E-mail: inbio@glas.apc.org

²Belozersky Institute of Physico-Chemical Biology, Lomonosov Moscow State University, Moscow, 119899 Russia; fax: (095) 939-3181

^* To whom correspondence should be addressed.

Received October 20, 1998; Revision received December 8, 1998

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It is known that ternary complexes of myosin subfragment 1 (S1) with ADP and the P_i analogs beryllium fluoride (BeF_x) and aluminum fluoride (AlF₄^-) are 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·BeF_x and S1·ADP·AlF₄^- in the absence and in the presence 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·BeF_x occurs much faster (3-4 min) than that of S1·ADP·AlF₄^- (hours). The formation of these complexes in the presence of F-actin led to dissociation of S1 from F-actin, this process being monitored by a decrease in light scattering. The light scattering decrease of the acto-S1 complex occurred much faster after addition of BeF_x (during 1 min) than after addition of AlF₄^- (more than 20 min). In both cases the light scattering of the acto-S1 complex decreased by 40-50%, but it remained much higher than that of F-actin measured in the absence of S1. The interaction of the S1·ADP·BeF_x and S1·ADP·AlF₄^- 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·BeF_x or S1·ADP·AlF₄^- to F-actin is accompanied by a fast increase in light scattering, but it does not affect the fluorescence of a pyrene label specifically attached to F-actin. We conclude from these data that within this time range a “weak” binding of the S1·ADP·BeF_x and S1·ADP·AlF₄^- complexes to F-actin occurs without the subsequent transition of the “weak” binding state to the “strong” binding state. Comparison of the light scattering kinetic curves shows that S1·ADP·AlF₄^- binds to F-actin faster than S1·ADP·BeF_x does: the second-order rate constants for the “weak” binding to F-actin are (62.8 ± 1.8)·10⁶ M^-1·sec^-1 in the case of S1·ADP·AlF₄^- and (22.6 ± 0.4)·10⁶ M^-1·sec^-1 in the case of S1·ADP·BeF_x. We conclude that the stable ternary complexes S1·ADP·BeF_x and S1·ADP·AlF₄^- can be successfully used for kinetic studies of the “weak” binding of the myosin heads to F-actin.

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KEY WORDS: myosin subfragment 1, F-actin, “weak” binding of myosin to actin, kinetics, skeletal muscle

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