Polymerization of vertebrate non-muscle and smooth muscle myosins.
We investigated how light chain phosphorylation controls the stability of filaments of vertebrate non-muscle myosins (from bovine thymocytes and chicken intestine epithelial brush border cells) and smooth muscle myosin (from chicken gizzard) in vitro. Using a sedimentation assay, the solubilities of the myosins were determined by measuring the amounts of myosin monomers (Cm) and filaments (Cp) present under a given set of conditions as a function of the total myosin concentration (Ct). Below 200 mM-NaCl, each myosin displayed distinct "critical monomer concentrations" (Cc) for polymerization, which were dependent on the salt concentration, the state of light chain phosphorylation and the presence of MgATP. At 150 mM-NaCl, MgATP increased the Cc of non-phosphorylated brush border myosin approximately five to tenfold, thymus myosin approximately 10 to 15-fold, and gizzard myosin approximately 25 to 50-fold. When these myosins were phosphorylated, MgATP had little effect on their solubilities, and their Cc values remained low. Analytical ultracentrifugation and electron microscopy demonstrated that the myosins were present in three different conformational states under the conditions used in the sedimentation assays, i.e. filaments, extended monomer (6 S) and folded monomer (10 S). Since at equilibrium only filaments and monomers were observed, we suggest that the polymerization pathway for these myosins can be analysed in terms of a dynamic monomer-polymer equilibrium (polymer in equilibrium 6 S monomer in equilibrium 10 S monomer). At roughly physiological ionic strength, light chain dephosphorylation (in the presence of MgATP) promotes the folded state (10 S), whereas phosphorylation promotes the extended state (6 S), and thereby favours filament assembly. The relevance of the monomer-polymer equilibrium to the state of organization of the myosin in vivo is discussed.
MRC Laboratory of Molecular Biology, Cambridge, U.K.
J
J
Kendrick-Jones
R C
RC
Smith
R
R
Craig
S
S
Citi
D000818:Animals; D002417:Cattle; D002645:Chickens; D005895:Gizzard, Avian; D008854:Microscopy, Electron; D008871:Microvilli / Q000032:analysis; D009130:Muscle, Smooth / Q000032:analysis*; D009218:Myosins; D010766:Phosphorylation; D011108:Polymers; D012995:Solubility; D013950:Thymus Gland; D014461:Ultracentrifugation
D011108:Polymers; D009218:Myosins