PROPERTIES OF THE NOVEL INTERMEDIATE FILAMENT PROTEIN SYNEMIN AND ITSIDENTIFICATION IN MAMMALIAN MUSCLE

We examined specific properties of highly purified synemin (230 kDa), recently identified as a novel intermediate filament (IF) protein, fro m avian smooth muscle. Soluble synemin in 10 mM Tris-HCl, pH 8.5, appe ars as similar to 11-nm-diameter globular structures by negative-stain and low-angle shadow electron microscopy. Chemical crosslinking and S DS-PAGE analysis indicate that soluble synemin molecules contain two 2 30-kDa subunits, The pH- and ionic strength-dependent solubility prope rties of synemin are similar to those of the type III IF protein desmi n, but under physiological-like conditions in which desmin self-assemb les into long similar to 10-nm-diameter IFs, synemin self-associates i nto complex, approx 15- to 25-nm-diameter globular structures. Calpain digestion demonstrated that synemin is extremely proteolytically labi le. Western blot analysis, with monospecific polyclonal antibodies aga inst avian synemin, shows the presence of the reactive 230-kDa synemin band in samples of adult avian skeletal, cardiac, and smooth muscle a nd of two reactive bands at similar to 225 kDa (major) and similar to 195 kDa in adult porcine skeletal, cardiac, and smooth muscle. Partial purification of synemin from porcine smooth muscle also resulted in f ractions highly enriched in the similar to 225- and similar to 195-kDa polypeptides, Conventional immunofluorescence and immunoconfocal micr oscopy of isolated myofibrils and of frozen sections also demonstrated , for the first time, that synemin is present in all three adult porci ne muscle cell types and is colocalized with desmin in skeletal and ca rdiac muscle cells at the myofibrillar Z-lines. (C) 1998 Academic Pres s.