Dictyostelium myosin: characterization of chymotryptic fragments and localization of the heavy-chain phosphorylation site.

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Abstract
Chymotrypsin cleaves Dictyostelium myosin in half, splitting the H chain (210,000 daltons) into 2 fragments of 105,000 daltons each. One of the 2 major fragments is soluble at low ionic strength and has a native MW of .apprx. 130,000. As judged by SDS [sodium dodecyl sulfate] polyacrylamide gel electrophoresis, this soluble fragment consists of the 2 intact myosin light chains of 18,000 and 16,000 daltons and a 105,000 dalton polypeptide derived from the myosin H chain. The soluble fragment retains actin-activated ATPase activity and the ability to bind to actin in an ATP-dissociable fashion. The maximal velocity of the actin-activated ATPase activity of the soluble fragment is 80% of that of uncleaved myosin; its apparent Km for actin is 12-fold > that of myosin. In addition to the major soluble 105,000 dalton fragment discussed above, chymotryptic cleavage of the Dictyostelium myosin generates fragments that are insoluble at low ionic strength. The major insoluble fragment is 105,000 daltons on an SDS polyacrylamide gel and forms thick filaments devoid of myosin heads. A less prevalent insoluble fragment has a MW of 83,000 and its probably a subfragment of the insoluble 105,000 dalton fragment. The H chain of myosin is phosphorylated in vivo and the phosphorylation site was localized to the insoluble fragments, which derive from the tail portion of the myosin molecule.