ACTIVATION OF REGULATED ACTIN BY SH1-MODIFIED MYOSIN SUBFRAGMENT-1

The reactive SH1 (Cys-707) group of the myosin subfragment 1 (S1) has been used frequently as an attachment site for fluorescent and spin pr obes in solution and muscle fiber experiments. In this study we examin ed (i) the motor function of SH1 spin-labeled heavy meromyosin (HMM) i n the in vitro motility assays and (ii) the effect of SH1-modified S1 on the motility of regulated actin, i.e., actin complexed with tropomy osin and troponin. N-ethylmaleimide (NEM), yl-2,2,6,6-tetramethyl-4-pi peridinyl)-iodacetamide (IASL), -[[(iodoacetyl)amino]ethyl]1-sulfo-5-n aphthylamine (IAEDANS), and iodoacetamide (IAA) were used to selective ly modify the SH1 group on S1; the SH1 group on HMM was labeled with I ASL, In the in vitro motility assays, 10-20% of unregulated actin fila ments moved at a speed of similar to 1 mu m/s over a surface coated wi th 90-95% modified IASL-HMM, Actin sliding was not observed with 95-98 % modified IASL-HMM, The sliding of regulated actin over unmodified HM M was activated by the addition of S1 modified with any of the SH1 rea gents to the in vitro motility assay solutions; both the speeds and th e percentage of the moving filaments increased at pCa 5, 7, and 8, To shed light on the activation of regulated actin sliding by SH1-modifed S1, acto-S1 ATPase and the binding to actin were determined for IASL- S1. While the binding affinities to actin were similar for IASL-S1 and unmodified S1 in the presence and absence of ADP and ATP, the K-m and V-max values were approximately 10-fold lower for the modified protei n. It is concluded that the activation of regulated actin by SH1-modif ed S1 facilitates the interaction of unmodified HMM heads with actin a nd thus can increase the sliding speeds and the percentage of regulate d actin filaments that move in the in vitro motility assays.