Covalent crosslinking of myosin subfragment-1 and heavy meromyosin to actin at various molar ratios: different correlations between ATPase activity and crosslinking extent.

This paper describes a systematic study of crosslinking of skeletal muscle myosin subfragment-1 (S1) and heavy meromyosin (HMM) to F-actin in the rigor state with 1-ethyl-3-[3-(dimethylamino)propyl]carbodiimide (EDC). We followed the time courses of S1 or HMM head crosslinking at various actin:S1 or actin:HMM head molar ratios and the resulting superactivation of ATPase activity. The ATPase activity of the covalent complexes was measured at 0.5 M KCl, where the covalent complexes retain superactivated ATPase activity but the activity of uncrosslinked myosin heads is not activated by actin. S1 crosslinking was slowest at the actin:S1 molar ratio of 1:1, but faster at larger molar ratios, where more than 80% of added S1 could be crosslinked to actin. In spite of the dependence of crosslinking rate on actin:S1 ratio, there were two linear correlations between ATPase activity and the extent of S1 crosslinking to actin: one for S1 crosslinked to actin at actin:S1 molar ratios more than 2.7:1 and the other for S1 crosslinked at a molar ratio of 1:1. Extrapolation of the former correlation line to 100% crosslinked S1 gave an ATPase activity of 39 s-1 for actin-S1 covalent complex at 25 degrees C, whereas that of the other correlation line gave 21 s-1. The latter smaller activity suggests that the interface between actin and S1 in their rigor complexes at a molar ratio of 1:1 is different from that at molar ratios of more than 2.7:1. The acto-HMM crosslinking rate depended on the ratio of actin to HMM head, like that of S1 crosslinking to actin. The ATPase activity of crosslinked actin-HMM was, unlike that of actin-S1 covalent complexes, bell-shaped as a function of the crosslinked heads, but chymotryptic conversion of HMM to S1 in the covalent complexes made the bell-shaped characteristics disappear and increased the activity close to that of actin-S1 covalent complexes. These results indicate that some physical constraint imposed on myosin heads suppresses the actin-activated ATPase activity of HMM crosslinked to actin.