MECHANISM OF MYOSIN SUBFRAGMENT-1-INDUCED ASSEMBLY OF CAG-ACTIN AND MGC-ACTIN INTO F-ACTIN-S-1-DECORATED FILAMENTS

The kinetics and mechanism of myosin subfragment-1-induced polymerizat ion of G-actin into F-actin-S-1-decorated filaments have been investig ated in low ionic strength buffer and in the absence of free ATP. The mechanism of assembly of F-actin-S-1 differs from salt-induced assembl y of F-actin. Initial condensation of G-actin and S-1 into oligomers i n reversible equilibrium is a prerequisite step in the formation of F- actin-S-1. Oligomers have a relatively low stability (10(6) M-1) and c ontain S-1 in a molar ratio to actin close to 0.5. Increased binding o f S-1 up to a 1:1 molar ratio to actin is associated with further irre versible condensation of oligomers into large F-actin-S-1 structures o f very high stability. In contrast to salt-induced assembly of F-actin , no monomer-polymer equilibrium, characterized by a critical concentr ation, can be defined for F-actin-S-1 assembly, and end-to-end anneali ng of oligomers is predominant over growth from nuclei in the kinetics . Simultaneous recordings of the changes in light scattering, pyrenyl- and NBD-actin fluorescence, ATP hydrolysis, and release of P-i during the polymerization process have been analyzed to propose a minimum ki netic scheme for assembly, within which several elementary steps, foll owing oligomer formation, are required for assembly of F-actin-S-1. AT P hydrolysis occurs before polymerization of MgATP-G-actin but not of CaATP-G-actin. The release of inorganic phosphate occurs on F-actin-S- 1 at the same rate as on F-actin.