The biochemical and structural properties of vertebrate thick filaments.

An extraction technique has been developed that removes 50% of the regulatory light chains (RLCs) from native vertebrate myosin filaments. The extraction susceptible population correlates with a chymotryptic resistant population which can be shown to be present. Evidence is provided which suggests that all myosin molecules in native thick filaments are identical and lose one of their two RLCs during the extraction procedure. RLC removal results in the exposure of a 'sticky patch' which interacts with the filament shaft sticking the myosin cross bridge down. The ionic strength dependance of this interaction suggests that it occurs via electrostatic and not hydrophobic residues. A two fold activation in the myosin ATPase activity can be demonstrated on the addition of calcium to purified native myosin filaments (PNFs). We demonstrate that the RLCs are intimately involved in the calcium sensitivity of the myosin ATPase. We show that after the removal of 50% of the RLCs a 50% reduction in the calcium sensitivity of the ATPase is observed suggesting an activatory role for the RLCs in vertebrate myosin. Hydrodynamic analysis reveals that PNFs possess a high affinity calcium binding site (Kd = 30?M) and also a low affinity non specific site. The binding of cations to these sites has a clear effect on the hydrodynamic properties which has been used to study the relationship between them. Such analysis reveals that the Kd of the low affinity site is modulated by calcium binding to the high affinity site. The significance of such interactions and their role in the contractile cycle is discussed.