A new technique for obtaining a myofibril‐like preparation from vertebrate smooth muscle has been developed. An actomyosin can be readily extracted from these myofibrils at low ionic strength and in yields 20 times as high as previously reported. The protein composition of all preparations has been monitored using dodecylsulfate‐gel electrophoresis. By this method smooth muscle actomyosin showed primarily only the major proteins, myosin, actin and tropomyosin, while the myofibrils contained, additionally, three new proteins not previously described with polypeptide chain weights of 60000, 110000 and 130000. The ATPase activities of both the myofibrils and actomyosin preparations are considerably higher than previously described for vertebrate smooth muscle. They are sensitive to micromolar Ca²⁺ ion concentrations to the same degree as comparable skeletal and cardiac muscle preparations, even though troponin‐like proteins could not be identified in these smooth muscle preparations. From the latter observation and the presence of Ca²⁺ ‐sensitivity in tropomyosin‐free actomyosin it is suggested that this calcium sensitivity is, as in some invertebrate muscles, a property of the myosin molecule.

Antibodies to chicken gizzard myosin, subfragment 1, light chain 20, and light meromyosin were used to visualize myosin in stress fibers of cultured chicken cells. The antibody specificity was tested on purified gizzard proteins and total cell lysates using immunogold silver staining on protein blots. Immunofluorescence on cultured chicken fibroblasts and epithelial cells exhibited a similar staining pattern of antibodies to total myosin, subfragment 1, and light chain 20, whereas the antibodies to light meromyosin showed a substantially different reaction. The electron microscopic distribution of these antibodies was investigated using the indirect and direct immunogold staining method on permeabilized and fixed cells. The indirect approach enabled us to describe the general distribution of myosin in stress fibers. Direct double immunogold labeling, however, provided more detailed information on the orientation of myosin molecules and their localization relative to alpha-actinin: alpha-actinin, identified with antibodies coupled to 10-nm gold, was concentrated in the dense bodies or electron-dense bands of stress fibers, whereas myosin was confined to the intervening electron-lucid regions. Depending on the antibodies used in combination with alpha-actinin, the intervening regions revealed a different staining pattern: antibodies to myosin (reactive with the head portion of nonmuscle myosin) and to light chain 20 (both coupled to 5-nm gold) labeled two opposite bands adjacent to alpha-actinin, and antibodies to light meromyosin (coupled to 5-nm gold) labeled a single central zone. Based on these results, we conclude that myosin in stress fibers is organized into bipolar filaments.

In vertebrate smooth muscle actomyosin and myofibrils a myosin light chain of molecular weight about 20000 becomes phosphorylated at the same Ca²⁺ concentration as required to stimulate the actin‐activated ATPase activity of myosin. Further, the degree of phosphorylation in the preparations as well as in various reconstituted actomyosins is proportional to their measured Ca²⁺ sensitivity. The phosphorylation process is very rapid and is essentially completed before the rise in ATPase activity. The enzyme responsible for the observed myosin phosphorylation is a specific myosin light chain kinase which is routinely co‐purified with myosin. This kinase is normally present in actomyosin and its removal together with tropomyosin leads to a complete loss of the actin‐activated ATPase activity. It is suggested that the Ca‐dependent phosphorylation of the light chain via the light chain kinase represents the initial step in the activation of myosin that leads to contraction. Relaxation is probably effected by an as yet uncharacterised light chain phosphatase.