Filaments with a diameter of 80-120 A have been prepared from 14-d-old chick embryonic skeletal muscle, using a physiological salt solution and gel filtration chromatography. The filaments obtained are composed of the two known muscle intermediate-filament proteins, vimentin and desmin, as well as the vimentin- and desmin-associated high molecular weight protein, synemin (230,000 mol. wt). In addition, they contain a previously unidentified high molecular weight protein (280,000 mol wt) which differs from synemin by isoelectric point, molecular weight, and immunological reactivity.

Immunofluorescence on cultured myogenic cells,using antisera to the 280,000-dalton polypeptide, has revealed that this protein has the same spatial distribution as desmin, vimentin, and synemin in both early myotubes, where it associates with cytoplasmic filaments, and late in myotubes, where it is associated with myofibril Z lines. Examination by immunofluorescence of frozen sections of developing embryonic skeletal muscle reveals a gradual diminution in the presence of the 280,000-dalton protein. The 280,000-dalton protein is undetectable in adult skeletal and smooth muscle, as shown by immunofluorescence and immunoautoradiography. In chick embryonic fibroblasts grown in tissue culture, only a subpopulation of the cells is reactive with antibodies to the 280,000-dalton protein even though all these cells contain vimentin. In the reactive cells, vimentin and the 280,000-dalton polypeptide exhibit an indistinguishable cytoplasmic filamentous network, which aggregates into filamentious bundles when the cells are exposed to colcemid. These results suggest that this newly identified high molecular weight protein is closely associated with intermediate filaments containing either vimentin alone or vimentin, desmin and synemin. The expression of this protein appears to be developmentally regulated and does not appear to parallel the expression of any of the other three intermediate-filament proteins. The absence of the 280,000-dalton polypeptide in adult muscle cells and its gradual reduction during development implies that is probably not required for the maintenance of Z-disk structure after the assembly of the sarcomere.

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