Ordered phosphorylation of the two 20,000 molecular weight light chains of smooth muscle myosin

Abstract

The time courses of phosphorylation of the MW 20,000 L chains by purified [rabbit] myosin L chain kinase plus calmodulin were determined. In confirmation of an earlier report, a steady-state kinetic analysis indicates that the phosphorylation occurs in an ordered manner; i.e., at a phosphorylation level of 0.5 mol of 32P incorporated/mol of bound MW 20,000 L chain, each myosin molecule would have 1 phosphorylated head. The kinetic parameters obtained for the phosphorylation of the more reactive myosin head are similar to those determined by using isolated L chains. The ordered, or sequential, phosphorylation and the different reactivities of the 2 MW 20,000 L chains, is evidently the result of preexisting asymmetry of the myosin molecule. Similar patterns of myosin phosphorylation are obtained in the absence and presence of skeletal muscle actin.