The Ca2+-dependent regulation of smooth muscle actomyosin involves a myosin light chain kinase (ATP: myosin light chain phosphotransferase). It has been shown (Dabrowska, R., Aromatorio, D., Sherry, J.M.F., and Hartshorne, D.J. 1977, Biochem. Biophys. Res. Commun. 78, 1263) that the kinase is composed of two proteins of approximate molecular weights 105 000 and 17 000. In this communication it is demonstrated that the 17 000 component is the modulator protein. This conclusion is based on: (1) the identical behavior of the 17 000 kinase component and modulator protein in assays of actomyosin Mg2+-ATPase activity, phosphorylation of myosin, and phosphodiesterase activity, and, (2) the similarity of the 17 000 kinase component and the modulator protein with respect to amino acid composition, absorption spectrum, and electrophoresis in urea-polyacrylamide gels. It is shown also that the modulator protein from smooth muscle and troponin C are distinct proteins.

In the theory of regulation of smooth-muscle actomyosin involving phosphorylation, it is generally accepted that the Mg2+-ATPase activity of phosphorylated myosin is activated by actin, whereas the activity of the unphosphorylated myosin is not. The role of Ca2+, however, is not clear and here there are basically two possibilities; i.e., either Ca2+ acts only to activate the myosin light-chain kinase or, that in addition to the sites on the myosin light-chain kinase, other regulatory sites are involved, presumably on the myosin molecule. To distinguish between these possibilities, three types of experiments were performed: (1) Gizzard myosin was phosphorylated and then separated from the myosin light-chain kinase and phosphatase by chromatography on Sepharose 4-B.

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