ORIENTATION CHANGES IN MYOSIN REGULATORY LIGHT-CHAINS FOLLOWING PHOTORELEASE OF ATP IN SKINNED MUSCLE-FIBERS

The orientation of the light-chain region of myosin heads in muscle fi bers was followed by polarized fluorescence from an extrinsic probe du ring tension transients elicited by photolysis of caged ATP. Regulator y light chain from chicken gizzard myosin was covalently modified with iodacetamidotetramethylrhodamine and exchanged into skinned fibers fr om rabbit psoas muscle without significant effect on the tension trans ients. fluorescence polarization ratios Q(parallel to) = ((parallel to )/(parallel to) - (perpendicular to)/(parallel to))/((parallel to)/(pa rallel to) + (perpendicular to)/(parallel to)) and Q(perpendicular to) = ((perpendicular to)/(perpendicular to) - (parallel to)/(perpendicul ar to))/((<perpendicular) (to>)/(perpendicular to) + (parallel to)/(pe rpendicular to)),where (m)/(n) denote fluorescence intensities for exc itation (pre-subscript) and emission (post-subscript) parallel or perp endicular to the fiber axis, were simultaneously measured at 0.5 ms ti me resolution. Q(perpendicular to) decreased and Q(parallel to) increa sed promptly after ATP release in the presence or absence of Ca2+, ind icating changes in orientation of the light-chain region associated wi th ATP binding or cross-bridge detachment. Little further change in th e Q signals accompanied either active tension development (+Ca2+) or t he final relaxation (-Ca2+). The Q and tension transients slowed when liberated ATP concentration was reduced. Assuming that ATP is released at 118 s(-1) (20 degrees C), the apparent second-order rate constants were 3-10 x 10(5) M(-1) s(-1) for Q(parallel to), 1-5 x 10(5) M(-1) s (-1) for Q(perpendicular to), and 0.5-2 x 10(5) M(-1) S-1 for the Conv ergence of tension traces starting from different rigor values, fittin g of model orientation distributions to the Q signals indicated that t he angular disorder increases after ATP binding. This orientation chan ge is specific to ATP because photorelease of ADP caused much smaller changes in the Q signals.