Universidade Federal de Uberlndia, Instituto de Gentica e Bioqumica, Bairro Umuarama, Uberlndia, Minas Gerais, Brazil.
Cell Biol Int. 2011 Mar;35(3):259-66. doi: 10.1042/CBI20090397.
Actomyosin precipitation is a critical step in the purification of myosins. In this work, the objective was to precipitate rat kidney actomyosin and isolate myosin by freezing and thawing the soluble fraction. Kidney was homogenized in imidazole buffer, centrifuged at 45000 g for 30 min, and the supernatant was frozen at -20°C for 48 h. The supernatant was thawed at 4°C, centrifuged at 45000 g for 30 min and the precipitate washed twice with imidazole buffer pH 7.0 (with and without Triton X-100, respectively). The resulting precipitate presented a polypeptide profile in SDS/PAGE characteristic of actomyosin and expressed Mg- and K/EDTA-ATPase activity. The actomyosin complex was solubilized with ATP and Mg, and the main polypeptide, p200, was purified in a DEAE-Sepharose column. p200 was marked with anti-myosin II, co-sedimented with F-actin in the absence, but not in the presence, of ATP and was identified by MS/MS with a high Mascot score for myosin IIA. The analysis identified peptides exclusive of myosin IIB, but detected no peptides exclusive of myosin IIC.
肌球蛋白的沉淀是肌球蛋白纯化的关键步骤。在这项工作中，目的是通过冷冻和解冻可溶性部分沉淀大鼠肾肌球蛋白并分离肌球蛋白。将肾组织在咪唑缓冲液中匀浆，以 45000g 离心 30 分钟，将上清液在-20°C 下冷冻 48 小时。将上清液在 4°C 下解冻，以 45000g 离心 30 分钟，并用咪唑缓冲液 pH7.0（分别用和不用 Triton X-100）洗涤沉淀两次。所得沉淀在 SDS/PAGE 中的多肽图谱特征为肌球蛋白和表达 Mg 和 K/EDTA-ATP 酶活性。用 ATP 和 Mg 将肌球蛋白复合物溶解，并在 DEAE-琼脂糖柱上纯化主要多肽 p200。p200 用抗肌球蛋白 II 标记，在没有 ATP 的情况下与 F-肌动蛋白共沉淀，但在有 ATP 的情况下不沉淀，并通过 MS/MS 鉴定为肌球蛋白 IIA 的高 Mascot 评分。分析鉴定了仅存在于肌球蛋白 IIA 中的肽，而未检测到仅存在于肌球蛋白 IIB 或肌球蛋白 IIC 中的肽。
