MAPPING PHOSPHORYLATION SITE OF THE REGULATORY LIGHT-CHAIN IN SMOOTH-MUSCLE MYOSIN BY IMMUNOELECTRON MICROSCOPY

Phosphorylation site responsible Ibr the regulation of smooth muscle m yosin was mapped using a polyclonal antibody against a phosphorylated hendecapeptide corresponding to the amino acid sequence around Ser-19 in the regulatory light chain. Phosphorylated myosin mixed with the an tibody was rotary-shadowed and was examined by electron microscopy. Th e antibody binding site was located in the head portion of myosin and the average distance from the head-rod junction was about 3 nm toward the tip of myosin head. The results indicate that the phosphorylated S er-19 in regulatory light chain is a little more extended toward the a djacent essential light chain in reference to the resolved N-terminal residues of the regulatory light chain in the three dimensional struct ure of myosin :leads from other sources, in which the structure of the N-terminal portions homologous to the phosphorylated Ser-19 was not r esolved (Rayment, I. et al. (1993) Science 261, 50-58; Xie, X. et al. (1994) Nature 368, 306-312). Intramolecular interaction through the in troduced phosphoryl group may be the primary results in the regulatory light chain which releases the motor domain from its suppressed state .