Direct visualization by electron microscopy of the weakly bound intermediates in the actomyosin adenosine triphosphatase cycle.

We used a novel stopped-flow/rapid-freezing machine to prepare the transient intermediates in the actin-myosin adenosine triphosphatase (ATPase) cycle for direct observation by electron microscopy. We focused on the low affinity complexes of myosin-adenosine triphosphate (ATP) and myosin-adenosine diphosphate (ADP)-Pi with actin filaments since the transition from these states to the high affinity actin-myosin-ADP and actin-myosin states is postulated to generate the molecular motion that drives muscle contraction and other types of cellular movements. After rapid freezing and metal replication of mixtures of myosin subfragment-1, actin filaments, and ATP, the structure of the weakly bound intermediates is indistinguishable from nucleotide-free rigor complexes. In particular, the average angle of attachment of the myosin head to the actin filament is approximately 40 degrees in both cases. At all stages in the ATPase cycle, the configuration of most of the myosin heads bound to actin filaments is similar, and the part of the myosin head preserved in freeze-fracture replicas does not tilt by more than a few degrees during the transition from the low affinity to high affinity states. In contrast, myosin heads chemically cross-linked to actin filaments differ in their attachment angles from ordered at 40 degrees without ATP to nearly random in the presence of ATP when viewed by negative staining (Craig, R., L.E. Greene, and E. Eisenberg. 1985. Proc. Natl. Acad. Sci. USA. 82:3247-3251, and confirmed here), freezing in vitreous ice (Applegate, D., and P. Flicker. 1987. J. Biol. Chem. 262:6856-6863), and in replicas of rapidly frozen samples. This suggests that many of the cross-linked heads in these preparations are dissociated from but tethered to the actin filaments in the presence of ATP. These observations suggest that the molecular motion produced by myosin and actin takes place with the myosin head at a point some distance from the actin binding site or does not involve a large change in the shape of the myosin head

Ca 2+

In contrast to previous studies, a new fluorescent method was used to accurately determine the Ca2+ concentration in test solutions used to activate skinned rat cardiac cells. This method used the calcium green-2 fluorescent indicator, which is shown to change its fluorescence over the Ca2+ range responsible for Ca2+ activation of force and ATPase. The dissociation constant ( K d) of calcium green-2 for Ca2+ was determined for three different Mg2+ concentrations in solutions similar to those used in the experiment. Increasing Mg2+ concentration from 1.0 to 8.0 mM had no significant effect on the Ca2+sensitivity of either force or actomyosin ATPase activity, in contrast to previous reported studies on force. The ATPase activity was activated at lower Ca2+ concentration than the force. The ratio (ATPase/force) is proportional to the dissociation rate of force-generating myosin cross bridges and decreased during Ca2+ activation. These findings are consistent with the hypothesis that cardiac muscle contraction is activated by a single Ca2+-specific binding site on troponin C