Simplified separation of myosin from rabbit liver.
Myosin was extracted from fresh rabbit liver using a solution with an ionic strength of 0.3 with two protease inhibitors and ATP. The myosin fraction was centrifuged at high speed in the presence of ATP and MgCl2. Its precipitate was dissolved in 0.6 M KCl, and the solution was treated with ammonium sulfate (30-60%). The fraction that precipitated was dissolved in 0.6 M KCl and put on a Sephacryl column. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) showed that the myosin obtained was 90% pure. Its molecular weight was 220,000 by gel electrophoresis; by Sephacryl S-300 column chromatography, it was found to be a complex at high ionic strengths. The protein had high ATPase activity, comparable to that of the myosin prepared by D. L. Brandon (1976, Eur. J. Biochem. 65, 139-146). Electron micrographs of our myosin looked like myosins from nonmuscle cells purified by other workers. Slow preparation gave poor yields of myosin with low ATPase activity and extra bands on SDS-PAGE. Rapid handling of fresh materials is essential for obtaining myosin of satisfactory quality. Our simple method saves time and reagents.
D000251:Adenosine Triphosphatases / Q000378:metabolism; D000818:Animals; D002850:Chromatography, Gel; D004591:Electrophoresis, Polyacrylamide Gel; D066298:In Vitro Techniques; D008099:Liver / Q000032:analysis*; D008970:Molecular Weight; D009218:Myosins / Q000302:isolation & purification*; D011189:Potassium Chloride / Q000494:pharmacology; D011506:Proteins / Q000032:analysis; D011817:Rabbits
D011506:Proteins; D011189:Potassium Chloride; D000251:Adenosine Triphosphatases; D009218:Myosins