Robin C. Smith

Calf thymus has been used as a source of vertebrate non-muscle myosin.Starting from 100 g of fresh tissue, between 5 and 15 mg of myosin were isolated to a level of purity greater than 90%.Electron microscopy showed that the purified protein comprises two globular heads attached to a fibrous rod.The myosin contains approximately 2 mol of heavy chain (Mr -200,-000), 2 mol of M, = 20,000 light chain, and 2 mol of M, = 16,000 light chain.In 0.6 M KCl, its ATPase activity is stimulated by EDTA or Ca2+ and inhibited by M&*.Evidence was obtained which suggests that the myosin is not contaminated by significant levels of vascular smooth muscle myosin, and it is probably derived predominantly from thymic lymphocytes.The M, = 20,000 light chain of thymus myosin can substitute for the regulatory light chain of scallop myosin and thereby resensitize desensitized scallop myofibrils, suggesting that it may function as a regulatory subunit in its parent myosin.Data are presented which demonstrate that phosphorylation of the Mr = 20,000 light chain regulates the interaction between thymus myosin and actin.A positive correlation between the level of light chain phosphorylation and the actin-activated MgATPase activity of thymus myosin was consistently observed.In the presence of MgATP, at -0.15 M NaCl and pH 7.0, phosphorylation of the M, = 20,000 light chain also controls the stability of thymus myosin filaments.These data suggest that phosphorylation of the M, = 20,000 "regulatory" myosin light chain may play an important role in controlling actomyosin-based motility in thymus cells.During the past decade, considerable progress has been made in characterizing myosins isolated from a variety of nonmuscle cells (1-4).This paper describes the isolation and characterization of myosin from calf thymus, which we selected as a source of vertebrate non-muscle myosin because: (i) large quantities of fresh material can be used to obtain sufficient myosin for detailed biochemical studies and (ii)

Myosins isolated from vertebrate smooth muscles and non-muscle cells such as lymphocytes and platelets contain regulatory light chains (Mr = 20000), which are phosphorylated by a Ca2+-calmodulin-dependent kinase and dephosphorylated by a Ca2+-insensitive phosphatase. Phosphorylation of the regulatory light chains of these myosins in vitro regulates not only their interactions with actin but also their assembly into filaments. Under approximately physiological conditions (0.15 M NaCl, pH 7.0) stoichiometric levels of Mg-ATP disassemble these non-phosphorylated myosin filaments into species with sedimentation coefficients (So20,w) of approximately 11S. Hydrodynamic and electron microscope observations have indicated that this 11S species is a monomer with a folded conformation (Trybus et al., Proc. natn. Acad. Sci. U.S.A. 79, 6151 (1982)). Rotary shadowing reveals that the tails of disassembled gizzard and thymus myosins are folded twice at two hinge points to form a folded three-segment structure. Phosphorylation of the regulatory light chains of these myosins causes these folded 11S molecules to unfold into the conventional extended monomeric form (6S), which is able to assemble into filaments. Thus in vitro these myosin filaments can be assembled or disassembled by phosphorylation or dephosphorylation of their light chains. Whether these results have any relevance to the situation within living non-muscle and smooth muscle cells remains to be established.