A SINGLE MYOSIN HEAD CAN BE CROSS-LINKED TO THE N-TERMINI OF 2 ADJACENT ACTIN MONOMERS

Myosin subfragment-1 (S1) can be cross-linked to two actin monomers by 1-ethyl-3-[3-(dimethylamino)-propyl]-carbodiimide only when F-actin i s in excess over S1. Electron micrographs of the covalent actin(2)-S1 complex showed that S1 was cross-linked to two adjacent monomers of th e same actin filament. Cross-linking experiments with pre-proteolyzed S1 derivatives in combination with a proteolytic dissection of the int act covalent actin(2)-S1 adduct (m = 265 kDa), revealed that two N-ter minal segments of actin (residues 1-28) were covalently attached to a single S1 molecule. One was cross-linked to either the 20-kDa or the 5 0-kDa heavy chain fragments of S1, and the other only to the 50-kDa re gion. The doubly cross-linked product was formed under physiological i onic strength with S1 or with reconstituted myosin filaments, regardle ss of the presence of ADP or the regulatory proteins, tropomyosin and troponin. Finally, we found that this cross-linking could also take pl ace within myofibrils in the rigor state. These results demonstrate th at under nonsaturating conditions, the actin-S1 interface encompasses a much larger region than that recently proposed for the nonphysiologi cal, fully saturated actin filaments.