Abstract
The binding of actin to myosin subfragment 1 (S1) has been shown to occur as a two-step reaction [Coates, Criddle & Geeves (1985) Biochem. J. 232, 351-356]. In the first step actin is weakly bound and the second step involves the complex isomerizing to a more tightly bound state. This isomerization can be followed specifically by monitoring the fluorescence of actin that has been covalently labelled with N-(pyren-1-yl)-iodoacetamide at Cys-374 [Geeves, Jeffries & Millar (1986) Biochemistry 25, 8454-8458]. We report here that the presence of nucleotides and nucleotide analogues affects the equilibrium between the strongly bound and weakly bound states (referred to as K2). In the presence of ATP, [gamma-thio]ATP or ADP and vanadate a value of approx. less than 10(-2) was estimated for K2. In the presence of PPi or ADP a value of approx. 2.3 or 10 respectively was obtained. An increase in KCl concentration or the presence of 40% ethylene glycol was found to decrease K2 in the presence of ADP. The data presented here are consistent with the two-step binding model proposed by Geeves, Goody & Gutfreund [(1984) J. Muscle Res. Cell Motil. 5, 351-361], where it was suggested that the transition between weakly bound and strongly bound states is closely associated with the force-generating event in whole muscle.