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Purification of desmin from adult mammalian skeletal muscle
Abstract:
A method has been 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 [Small & Sobieszek (1977) J. Cell Sci. 23, 243-268]. The desmin was extracted in 1 M-acetic acid/20 mM-NaCl at 4 degrees C for 15h from the residue remaining after actomyosin extraction from washed myofibrils. Successive chromatography on hydroxyapatite and DEAE-Sepharose CL-6B in 6M-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 10mM-Tris/acetate (pH 8.5)/1 mM dithioerythritol and subsequent clarification at 134 000 g (rav. 5.9 cm) 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 degrees C resulted in the formation of synthetic desmin filaments have an average diameter of 9-11.5 nm. The present studies demonstrate that 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 10nm filaments have not been 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.
Insights
Researchers developed a method to purify desmin, a key protein in muscle, from porcine skeletal muscle. This purified desmin can form 10 nm filaments in vitro, supporting its potential cytoskeletal role in muscle cells.
Area of Science:
- Biochemistry
- Cell Biology
- Muscle Physiology
Background:
- Desmin is an intermediate filament protein crucial for muscle structure and function.
- Previous methods for desmin isolation were not optimized for skeletal muscle.
- The presence and function of desmin filaments in mature skeletal muscle remain under investigation.
Purpose of the Study:
- To develop a robust method for purifying desmin from mature mammalian skeletal muscle.
- To characterize the purity and properties of the isolated desmin.
- To investigate the in vitro filament-forming capacity of purified skeletal muscle desmin.
Main Methods:
- Extraction of desmin from washed myofibril residue after actomyosin removal.
- Purification using hydroxyapatite and DEAE-Sepharose CL-6B chromatography in 6M-urea.
- Urea removal by dialysis and clarification via ultracentrifugation.
- In vitro filament formation by dialysis against a specific buffer.
Main Results:
- A purification method yielding desmin >97% pure (55,000-dalton protein) with no detectable actin contamination.
- Obtained a clear desmin solution after urea removal and clarification.
- Purified desmin formed synthetic filaments with an average diameter of 9-11.5 nm under specific ionic conditions.
Conclusions:
- The developed method allows for isolation of sufficient quantities of pure desmin from skeletal muscle for detailed studies.
- The in vitro formation of 10 nm filaments by purified skeletal muscle desmin supports their potential existence and cytoskeletal role in vivo.
- Further research is warranted to unequivocally demonstrate desmin filaments in mature muscle cells.