Localization of the active site and phosphorylation site of Acanthamoeba myosins IA and IB

The 130- and 125-kDa heavy chains of Acanthamoeba myosins IA and IB were radioactively labeled at either the regulatory phosphorylation site or the catalytic site and then subjected to controlled proteolysis by either trypsin or chymotrypsin. The labeled and unlabeled peptides generated during the course of proteolysis were identified by autoradiography and Coomassie Blue staining after separation by electrophoresis on sodium dodecyl sulfate-polyacrylamide gels. The relative positions of the phosphorylation and active sites could be deduced. The catalytic site of myosin IA is most probably within 38 kDa of one end of the 130-kDa heavy chain, and the phosphorylation site, which can be no more than 40 kDa away from the catalytic site, would then be between 38 and 78 kDa of that same end of the heavy chain. Possibly, the phosphorylation site is further restricted to the region between 38 and 64 kDa from the end of the heavy chain. The catalytic and phosphorylation sites of myosin IB are both contained within a segment of 62 kDa at one end of the 125-kDa heavy chain and are within 40 kDa of each other. The phosphorylation site may be restricted to a small segment between 60 and 62 kDa from one end of the heavy chain which would limit the possible position of the catalytic site to the region between 20 and 60 kDa of that end.