Selective purification of the 20,000-Da light chains of smooth muscle myosin

• Published in: Analytical biochemistry Vol. 135; no. 1; p. 37
• Main Authors: Hathaway, D R, Haeberle, J R
• Format: Journal Article
• Language: English
• Published: United States 01-11-1983
• Subjects: Animals; Chickens; Chromatography - methods; Gizzard, Avian - analysis; Muscle, Smooth - analysis; Myosins - isolation & purification; Turkeys
• ISSN: 0003-2697
• DOI: 10.1016/0003-2697(83)90726-1

The 20,000-Da light chains of gizzard smooth muscle myosin have been purified to homogeneity. Actomyosin, prepared by MgATP extraction of myofibrils, was denatured in 8 M urea, 1 M guanidine HCl, and 0.05% sodium dodecyl sulfate. Myosin heavy chains were precipitated with ethanol and the light chain enriched fraction was dialyzed and subjected to chromatography on DEAE-Sephacel. Fractions containing the 20,000-Da light chains were further purified by hydrophobic chromatography on phenyl-Sepharose. The 20,000-Da light chains eluted at low ionic strength from the phenyl-Sepharose column were judged to be greater than 95% pure by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and contained only 0.04 mol of phosphate/mol of light chain. The yield of light chains was calculated to be 219 +/- 17 mg/kg of starting gizzard smooth muscle. This method may be useful for preparation of homogeneous 20,000-Da smooth muscle myosin light chains in the quantities necessary for study of contractile systems.