A NOVEL ELECTRON-PARAMAGNETIC-RESONANCE SPIN-LABEL AND ITS APPLICATION TO STUDY THE CROSS-BRIDGE CYCLE

We have used a novel alpha-iodoketone spin-label (IKSL) to study myosi n head orientation and cross-bridge dynamics in the putative pre-power stroke state. Possible perturbation of the cross-bridge cycle by the l abel was assayed by the sinusoidal analysis method (Kawai and Brandt, 1980; Kawai and Zhao, 1993), which determines the rate constants of th e elementary steps in the cycle. A comparison of the rates obtained fr om unlabeled and IKSL fibers revealed small (10-20%) changes in the AT P hydrolysis rate and in the rate constants of the elementary steps. T he labeling induced small changes (<13%) in the distribution of the cr oss-bridges among the various intermediate states. Pre-powerstroke cro ss-bridges were induced by aluminum fluoride in the presence of Ca2+ a nd ATP. In this state, force development is inhibited, but a large pro portion (40%) of the cross-bridges are still attached to the thin fila ment. We have used conventional electron paramagnetic resonance to mea sure orientation, and have found that the pre-powerstroke heads are as disordered as in relaxation. Their mobility, as determined by saturat ion transfer electron paramagnetic resonance, was significantly restri cted. Assuming that stiffness is proportional to the fraction of stron gly attached heads, the rotational correlation time of the bound heads is estimated to be tau(r) = similar to 150-400 mu s.