Purification of the intermediate filament-associated protein, synemin, from chicken smooth muscle. Studies on its physicochemical properties, interaction with desmin, and phosphorylation.

Synemin, a 230,000-dalton protein associated with desmin- and vimentin-containing intermediate filaments (Granger, B. L., and Lazarides, E. (1980) Cell 22, 727-738), has been purified from gizzard smooth muscle and biochemically characterized. Purification was achieved by extracting the salt-insoluble pellet of muscle protein with 6 M urea and chromatography of the urea extract on columns of hydroxylapatite, DEAE-Sephacel, and phosphocellulose. The soluble form of synemin is a globular tetramer of 980,000 daltons with a S20,w of 22.4 +/- 3.2. Synemin has a pI of 5.34, in agreement with its high content in glutamic acid (20%), and is rich in serine (11%) and poor in cysteine (0.4%). Synemin is phosphorylated in smooth muscle and is one of the muscle proteins with the highest capacity to incorporate exogenously added [32P]phosphate. Of the [32P] phosphate incorporated into synemin, 95% is bound to serine and only 5% to threonine. The phosphorylation of synemin is enhanced by the cyclic AMP analog, 8-Br-cyclic AMP. Immunofluorescence studies using anti-synemin antibodies show that purified synemin binds to filaments of desmin assembled in vitro. Synemin specifically inhibits the immunoprecipitation of purified soluble desmin by anti-desmin antibodies, indicating that synemin interacts in vitro with soluble desmin.