Purification of desmin from adult mammalian skeletal muscle

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Abstract
A method was developed for preparation of purified desmin from mature mammalian (porcine) skeletal muscle. A crude desmin-containing fraction was prepared by modification of procedures used for isolation of smooth-muscle intermediate, filament protein. The desmin was extracted in 1 M-acetic acid/20 mM-NaCl at 4.degree. C for 15 h from the residue remaining after actomyosin extraction from washed myofibrils. Successive chromatography on hydroxyapatite and DEAE[diethylaminoethanol]-Sepharose CL-6B in 6 M-urea yielded desmin that was routinely more than 97% 55,000 dalton protein and that had no detectable actin contamination. Removal of urea by dialysis against 10 mM-Tris/acetate (pH 8.5)/1 mM dithioerythritol and subsequent clarification at 134,000 g for 1 h results in a clear desmin solution. Dialysis of purified desmin against 100 mM-NaCl/1 mM-MgCl2/10 mM-imidazole/HCl, pH 7.0, at 2.degree. C resulted in the formation of synthetic desmin filaments having an average diameter of 9-11.5 nm. The relatively small amount of desmin in mature skeletal muscle can be isolated in sufficient quantity and purity to permit detailed studies of its properties and function. Although 10 nm filaments were not unequivocally demonstrated in mature muscle in vivo, that the purified skeletal-muscle desmin will form 10 nm filaments in vitro lends support to their possible existence and cytoskeletal function in mature skeletal-muscle cells.