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 fibers was followed by polarized fluorescence from an extrinsic probe during tension transients elicited by photolysis of caged ATP. Regulatory light chain from chicken gizzard myosin was covalently modified with iodacetamidotetramethylrhodamine and exchanged into skinned fibers from rabbit psoas muscle without significant effect on the tension transients. fluorescence polarization ratios Q(parallel to) = ((parallel to)/(parallel to) - (perpendicular to)/(parallel to))/((parallel to)/(parallel to) + (perpendicular to)/(parallel to)) and Q(perpendicular to) = ((perpendicular to)/(perpendicular to) - (parallel to)/(perpendicular to))/((<perpendicular) (to>)/(perpendicular to) + (parallel to)/(perpendicular to)),where (m)/(n) denote fluorescence intensities for excitation (pre-subscript) and emission (post-subscript) parallel or perpendicular to the fiber axis, were simultaneously measured at 0.5 ms time resolution. Q(perpendicular to) decreased and Q(parallel to) increased promptly after ATP release in the presence or absence of Ca2+, indicating changes in orientation of the light-chain region associated with ATP binding or cross-bridge detachment. Little further change in the Q signals accompanied either active tension development (+Ca2+) or the 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 Convergence of tension traces starting from different rigor values, fitting of model orientation distributions to the Q signals indicated that the angular disorder increases after ATP binding. This orientation change is specific to ATP because photorelease of ADP caused much smaller changes in the Q signals.