K Park, N Tatsumi, K Hashimoto, K Okuda, S Yamamoto
{"title":"Simplified separation of myosin from rabbit liver.","authors":"K Park, N Tatsumi, K Hashimoto, K Okuda, S Yamamoto","doi":"","DOIUrl":null,"url":null,"abstract":"<p><p>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.</p>","PeriodicalId":14978,"journal":{"name":"Journal of applied biochemistry","volume":null,"pages":null},"PeriodicalIF":0.0000,"publicationDate":"1985-08-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":"0","resultStr":null,"platform":"Semanticscholar","paperid":null,"PeriodicalName":"Journal of applied biochemistry","FirstCategoryId":"1085","ListUrlMain":"","RegionNum":0,"RegionCategory":null,"ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":null,"EPubDate":"","PubModel":"","JCR":"","JCRName":"","Score":null,"Total":0}
引用次数: 0
Abstract
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.
采用离子强度为0.3的溶液，加入两种蛋白酶抑制剂和ATP，从新鲜兔肝中提取肌球蛋白。肌球蛋白部分在ATP和MgCl2存在下高速离心。将其沉淀物溶解于0.6 M KCl中，用硫酸铵(30-60%)处理。将析出的馏分溶解于0.6 M KCl中，置于头孢丙烯柱上。十二烷基硫酸钠-聚丙烯酰胺凝胶电泳(SDS-PAGE)结果表明，所得肌球蛋白纯度为90%。凝胶电泳测定其分子量为22万;经sepphacryl S-300柱层析，发现其为高离子强度的配合物。该蛋白具有高atp酶活性，可与D. L. Brandon(1976，欧洲)制备的肌球蛋白相媲美。中国生物医学工程学报，2016,33(2):444 - 444。我们的肌凝蛋白的电子显微照片看起来像其他工人从非肌肉细胞纯化的肌凝蛋白。缓慢的制备导致肌球蛋白产量低，atp酶活性低，SDS-PAGE上有额外的条带。新鲜原料的快速处理是获得满意质量肌球蛋白的关键。我们的简单方法节省了时间和试剂。
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