Kinetic studies of the cooperative binding of subfragment 1 to regulated actin.

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Abstract
The transient-state kinetics of binding of myosin subfragment 1 (SF-1) to regulated actin in the presence and absence of Ca2+ were investigated [in the rabbit]. The binding of SF-1 to pure actin, to actin-tropomyosin (actin-TM) or to actin-tropomyosin-troponin (actin-TM-TN) in the presence of Ca2+ was kinetically the same. In each case, the light-scattering transients were biphasic, suggesting a 2-step binding of SF-1 to actin. Binding of SF-1 to regulated actin in the absence of Ca2+ differed from binding in its presence and varied depending on whether SF-1 or regulated actin was in excess. The kinetic results in the absence of Ca2+ are explained by a cooperative binding model, in which the initial binding of SF-1 molecules to open (active) actin sites increases the number of open sites. TN-I labeled with the fluorophore 4-(N-iodoacetoxyethyl-N-methyl)-7-nitrobenz-2-oxa-1,3 diazole (TN*) was used to probe the state of the actin-TM-TN complex. Binding of SF-1 or Ca2+ to regulated actin (in the absence of Ca2+) decreased the fluorescence of actin-TM-TN* by 30%, suggesting that binding of SF-1 or Ca2:+ induces a similar change in state. The change in fluorescence of TN* was used to measure the rate of the transition from the active to the relaxed state in the absence of Ca2+, which was 430 s-1 at 4.degree. C in 0.1 M KCl. The lag prior to association of SF-1 with regulated actin (in the absence of Ca2+) was abolished when 3 SF-1 molecules were prebound/7 G-actin monomers. A titration of actin-TM-TN* (in the absence of Ca2+) with SF-1 or SF-1-ADP showed that most actin sites are open, as measured by the fluorescence change, when the occupancy of actin-TM-TN* by SF-1-ADP or SF-1 is approximately 50%. The evidence shows that partial occupancy of a block of G-actin sites (possibly 7) by SF-1 or SF-1-ADP stabilizes the open (active) conformation.