Does phosphate release limit the ATPases of soleus myofibrils? Evidence that (A)M· ADP·Pi states predominate on the cross-bridge cycle

The ATPases (±Ca^sup 2+^) of myofibrils from rabbit soleus (a slow muscle) and psoas (a fast muscle) have different E^sub a^: -Ca^sup 2+^, 78 and 60 kJ/mol and +Ca^sup 2+^, 155 and 71 kJ/mol, respectively. At physiological temperatures, the two types of myofibrillar ATPase are very similar and yet the mechanical properties of the muscles are different (Candau et al. (2003) Biophys J 85: 3132-3141). Muscle contraction relies on specific interactions of the different chemical states on the myosin head ATPase pathway with the thin filament. An explanation for the E^sub a^ data is that different states populate the pathways of the two types of myofibril because the rate limiting steps are different. Here, we put this to the test by a comparison of the transient kinetics of the initial steps of the ATPases of the two types of myofibril at 4°C. We used two methods: rapid flow quench (`cold ATP chase': titration of active sites, ATP binding kinetics, k^sub cat^; `P^sub i^ burst': ATP cleavage kinetics) and fluorescence stopped-flow (MDCC-phosphate binding protein for free P^sub i^; myofibrillar tryptophan fluorescence for myosin head-thin filament detachment and ATP cleavage kinetics). We find that, as with psoas myofibrils, the most populated state on the cross-bridge cycle of soleus myofibrils, whether relaxed or activated, is (A)M·ADP·P^sub i^. We propose a reaction pathway that includes several (A)M·ADP·P^sub i^ sub-states that are either `weak' or `strong', depending on the mechanical condition.[PUBLICATION ABSTRACT]