Penn Medicine
   
 

Yale Goldman, M.D., Ph.D.

Orientation changes in myosin regulatory light chains following photorelease of ATP in skinned muscle fibers.

Allen TStC, Ling N, Irving M, Goldman YE

Biophys. J. 70:1847-1862, 1996.

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 (RLC) from chicken gizzard myosin was covalently modified with iodoacetamido tetramethylrhodamine (iodo-ATR) and exchanged into skinned fibers from rabbit psoas muscle without significant effect on the tension transients. Fluorescence polarization ratios Q|| = (||I|| - ^I||)/(||I|| + ^I||) and Q^ = (^I^ - ||I^)/(^I^ + ||I^), where mIn 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^ decreased and Q|| 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 oC), the apparent second order rate constants were 3 - 10 x 105 M-1s-1 for Q|| , 1 - 5 x 105 M-1s-1 for Q^, and 0.5 - 2 x 105 M-1s-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 following ATP binding. This orientation change is specific to ATP because photorelease of ADP caused much smaller changes in the Q signals.

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