Changes in the state of actin during superprecipitation of actomyosin

Exchangeability of actin-bound ADP and calcium in actomyosin at low ionic strength has been studied using F-actin labelled with [14C]ADP or 45Ca and measuring release of radioactivity into solution. Low-speed centrifugation, ultracentrifugation and ultrafiltration were used to separate protein from the medium. Comparison of the results obtained with these three separation procedures has revealed that the release of [14C]ADP and 45Ca into the medium in the presence of millimolar concentrations of MgATP is largely due to the release under these conditions of actin itself retaining its bound ADP and calcium. The real exchange of the bound nucleotide and calcium, even under the most favourable conditions, was in our experiments limited to about 20%. Detailed examination of the dependence of both the release of actin and the exchange of actin-bound ADP and calcium on the free divalent cations present, the kind and concentration of the added nucleotide, and temperature of incubation indicates that there is no correlation between the exchange and superprecipitation of actomyosin. The results presented support the view that the limited enhancement by myosin of the exchange of nucleotide and cation bound to actin under certain conditions results from accidental disruption of bonds between actin monomers due to a mechanical stress exerted on actin filaments upon their interaction with myosin filaments.