An Attempt to Detect Phosphorylated Myosin by ADP-ATP Exchange

For the purpose of detection of the intermediate E—OP in the reaction of myosin-ATPase [EC 3.6.1.3], the possibility of back-incorporation of 14C from labelled ADP into ATP was examined. The main project was to confirm the reverse reaction, ADP+E—OP↑ATP+E, by deceleration of the hydrolytic process, E-OP↑E+P1, with D2O. 2. Myosin-ATPase was incubated at 25°C with ATP and 14C-labelled ADP in the medium of D2O (pD 8.0). The mixture of the reaction products was desalted by a Sephadex G-10 treatment, and chromatographed on Dowex 1-X4, yielding three nucleotide fractions, i.e., AMP, ADP and ATP. The specific activities of three nucleotide fractions were calculated based on the radioactivities of these fractions determined by a scintillation counter. 3. After a prolonged incubation, a small amount of 14C was incorporated from ADP into ATP. However, an additional experiment showed that this back-incorporation was explainable as due to the action of myokinase [adenylate kinase, EC 2.7. 4.3], which was a contaminant in the myosin preparation. Thus a phosphorylated myosin, E-OP, is not demonstrable by this method.