We report the immunological characterization of the subunit of the intermediate sized (100 A) filaments from muscle cells. The protein as isolated from smooth muscle (chicken gizzard) has an apparent molecular weight of 50,000. It is insoluble in buffers that solubilize myosin and the majority of actin, but becomes soluble in the presence of urea. Under a variety of experimental conditions, that include the presence of 8 M urea, this new protein comigrates with actin during purification studies. The two proteins can be separated from each other by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate, and antibodies have been elicited against the 50,000 dalton protein purified by using this technique. These antibodies crossreact with the partially purified protein in urea, but show no detectable cross reaction with actin or myosin. Indirect immunofluorescence reveals that in skeletal muscle this protein is found in close association with the Z lines of the sarcomeres and extends between the Z lines of adjacent myofibrils; it is also associated with filamentous structures that run along the length of a muscle fiber both in close association with the plasma membrane and between myofibrils. These filaments appear to connect myofibrils to each other or to the plasma membrane at the level of their Z lines. In heart muscle, the protein shows the same distribution as in skeletal muscle. In addition, it is found intimately associated with intercalated disks and areas of membrane interaction between laterally associated heart muscle cells. The ). The cytoplasm of both developing and adult smooth muscle cells contains a great number of these filaments which comprise a class distinct from the actin and myosin filaments that are involved in cellular contraction (12, 13). These filaments are known to insert characteristically into desmosome-like structures associated with membrane which are somehow involved in maintaining a strong cell to cell interaction between smooth muscle cells (14). We used smooth muscle as a representative differentiated cell system for the isolation and characterization of the subunit of the 100 A filaments. In this paper, we report its immunological characterization from this type of muscle, and use indirect immunofluorescence to probe its localization in smooth, skeletal, and cardiac muscle cells. MATERIALS AND METHODS Antibody Preparation. Analytical and preparative slab gel electrophoresis with 12.5% gels was performed according to the discontinuous Tris-glycine system of Laemmli (15). The 50,000 molecular weight protein (desmin), for immunization, was purified from the 8 M urea extract of smooth muscle (see legend to Fig. if), by introducing as a final purification step preparative sodium dodecyl sulfate (NaDodSO4)/slab gel electrophoresis (refs. 16 and 17; see Results). After preparative electrophoresis of the 8 M urea extract (Fig. if), the gels were stained for 15 min in 0.25% Coomassie brilliant blue, made up in 50% methanol (vol/vol) and 7.5% acetic acid (...
ABSTRACFDesmin is a 50,000-mol wt protein that is enriched along with 100-/~ filaments in chicken gizzard that has been extracted with 1 M KI. Although 1 M KI removes most of the actin from gizzard, a small fraction of this protein remains persistently insoluble, along with desmin. The solubility properties of this actin are the same as for desmin: they are both insoluble in high salt concentrations, but are solubilized at low pH or by agents that dissociate hydrophobic bonds. Desmin may be purified by repeated cycles of solubilization by 1 M acetic acid and subsequent precipitation by neutralization to pH 4. During this process, a constant nonstoichiometric ratio of actin to desmin is attained.Gel filtration on Ultrogel AcA34 in the presence of 0.5% Sarkosyl NL-97 reveals nonmonomeric fractions of actin and desmin that comigrate through the column. Gel filtration on Bio-Gel P300 in the presence of 1 M acetic acid reveals that the majority of desmin is monomeric under these conditions. A small fraction of desmin and all of the actin elute with the excluded volume. When the acetic acid is removed from actin-desmin solutions by dial~,sis, a gel forms that is composed of filaments with diameters of 120-140 A. These filaments react uniformly with both anti-actin and anti-desmin antiserum. These results suggest that desmin is the major subunit of the muscle 100-/~ filaments and that it may form nonstoichiometric complexes with actin.
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