Hirabayashi T, Tamura R, Mitsui I, Watanabe Y
J Biochem. 1983 Feb;93(2):461-8. doi: 10.1093/oxfordjournals.jbchem.a134200.
Total protein constituents of Tetrahymena thermophila strain B1868 III were studied by two-dimensional agarose-polyacrylamide gel electrophoresis to detect actin among the constituents. In the attempts to prepare a whole-cell extract of Tetrahymena, it was found that protease activity in the extract was so high that high molecular components were quickly digested with the endogenous protease into small peptides unless the homogenization and heat-treatment in a sodium dodecylsulfate solution were performed within 5 s. It was eventually found that employment of 8 M guanidine hydrochloride (HCl) in the homogenization of cells perfectly prevented the degradation of protein components, even through a long preparation procedure. A devised two-dimensional agarose-polyacrylamide gel electrophoresis of the guanidine HCl extract gave a 'protein map' on which most proteins were located in their respective positions, including proteins with more than 200,000 mol. wt. Addition of rabbit skeletal muscle actin on the protein map revealed that no protein with isoelectric point and molecular weight identical with those of the actin was contained in the whole Tetrahymena extract, suggesting that Tetrahymena actin may have characteristics far different from those of skeletal muscle actin.
通过二维琼脂糖-聚丙烯酰胺凝胶电泳研究嗜热四膜虫B1868 III株的总蛋白质成分，以检测其中的肌动蛋白。在制备四膜虫全细胞提取物的过程中，发现提取物中的蛋白酶活性非常高，以至于除非在5秒内进行十二烷基硫酸钠溶液中的匀浆和热处理，否则高分子量成分会被内源性蛋白酶迅速消化成小肽。最终发现，在细胞匀浆中使用8 M盐酸胍（HCl）能完美地防止蛋白质成分的降解，即使经过长时间的制备过程。对盐酸胍提取物进行的二维琼脂糖-聚丙烯酰胺凝胶电泳得到了一张“蛋白质图谱”，大多数蛋白质都位于各自的位置，包括分子量超过200,000的蛋白质。在蛋白质图谱上添加兔骨骼肌肌动蛋白后发现，四膜虫全细胞提取物中不含有等电点和分子量与肌动蛋白相同的蛋白质，这表明四膜虫肌动蛋白可能具有与骨骼肌肌动蛋白截然不同的特性。
