Properties of the novel intermediate filament protein synemin and its identification in mammalian muscle.
We examined specific properties of highly purified synemin (230 kDa), recently identified as a novel intermediate filament (IF) protein, from avian smooth muscle. Soluble synemin in 10 mM Tris-HCl, pH 8.5, appears as approximately 11-nm-diameter globular structures by negative-stain and low-angle shadow electron microscopy. Chemical crosslinking and SDS-PAGE analysis indicate that soluble synemin molecules contain two 230-kDa subunits. The pH- and ionic strength-dependent solubility properties of synemin are similar to those of the type III IF protein desmin, but under physiological-like conditions in which desmin self-assembles into long approximately 10-nm-diameter IFs, synemin self-associates into complex, approx 15- to 25-nm-diameter globular structures. Calpain digestion demonstrated that synemin is extremely proteolytically labile. Western blot analysis, with monospecific polyclonal antibodies against avian synemin, shows the presence of the reactive 230-kDa synemin band in samples of adult avian skeletal, cardiac, and smooth muscle and of two reactive bands at approximately 225 kDa (major) and approximately 195 kDa in adult porcine skeletal, cardiac, and smooth muscle. Partial purification of synemin from porcine smooth muscle also resulted in fractions highly enriched in the approximately 225- and approximately 195-kDa polypeptides. Conventional immunofluorescence and immunoconfocal microscopy of isolated myofibrils and of frozen sections also demonstrated, for the first time, that synemin is present in all three adult porcine muscle cell types and is colocalized with desmin in skeletal and cardiac muscle cells at the myofibrillar Z-lines.
Bilak
S R
SR
Department of Animal Science, Iowa State University, Ames, Iowa, 50011-3260, USA.
Sernett
S W
SW
Bilak
M M
MM
Bellin
R M
RM
Stromer
M H
MH
Huiatt
T W
TW
Robson
R M
RM
D000818:Animals; D002154:Calpain; D002645:Chickens; D019084:Fluorescent Antibody Technique, Indirect; D006863:Hydrogen-Ion Concentration; D007381:Intermediate Filament Proteins / Q000737:chemistry* / Q000302:isolation & purification / Q000648:ultrastructure; D008854:Microscopy, Electron; D008970:Molecular Weight; D009124:Muscle Proteins / Q000737:chemistry* / Q000302:isolation & purification / Q000648:ultrastructure; D009132:Muscles / Q000737:chemistry*; D009994:Osmolar Concentration; D011487:Protein Conformation; D013552:Swine
D007381:Intermediate Filament Proteins; D009124:Muscle Proteins; C430456:desmuslin; D002154:Calpain