The ADP release step of the smooth muscle cross-bridge cycle is not directly associated with force generation

When smooth muscle myosin subfragment I (S1) is bound to actin filaments in vitro, the light chain domain tilts upon release of MgADP, producing a sim ilar to 3.5-nm axial motion of the head-rod junction (Whittaker et al., 199 5. Nature. 378:748-751). If this motion contributes significantly to the po wer stroke, rigor tension of smooth muscle should decrease substantially in response to cross-bridge binding of MgADP. To test this prediction, we mon itored mechanical properties of permeabilized strips of chicken gizzard mus cle in rigor and in the presence of MgADP. For comparison, we also tested p soas and soleus muscle fibers. Any residual bound ADP was minimized by incu bation in Mg2+-free rigor solution containing 15 mM EDTA. The addition of 2 mM MgADP, while keeping ionic strength and free Mg2+ concentration constan t, resulted in a slight increase in rigor tension in both gizzard and soleu s muscles, but a decrease in psoas muscle. In-phase stiffness monitored dur ing small (<0.1%) 500-Hz sinusoidal length oscillations decreased in all th ree muscle types when MgADP was added. The changes in force and stiffness w ith the addition of MgADP were similar at ionic strengths from 50 to 200 mM and were reversible. The results with gizzard muscle were similar after th iophosphorylation of the regulatory light chain of myosin. These results su ggest that the axial motion of smooth muscle S1 bound to actin, upon dissoc iation of MgADP, is not associated with force generation. The difference be tween the present mechanical data and previous structural studies of smooth S1 maybe explained if geometrical constraints of the intact contractile fi lament array alter the motions of the myosin heads.