Avissar N, de Vries A, Ben-Shaul Y, Cohen I
Biochim Biophys Acta. 1975 Jan 14;375(1):35-43. doi: 10.1016/0005-2736(75)90070-x.
A fibrillar protein complex, possessing ouabain-insensitive Ca2+-ATPase activity was isolated from human erythrocyte membranes by using a low ionic strength extraction procedure. Mg2+-ATPase activity was revealed upon addition of rabbit skeletal muscle actin, thus demonstrating the presence of a myosin-like protein in the crude extract of the erythrocyte membrane. Upon sodium dodecylsulfate gel electrophoresis, the extract showed mainly the doublet of subunit molecular weight bands of 230 000 and 210 000, and more than 10 faster moving bands. Gel filtration of the erythrocyte membrane extract on Sepharose 4B furnished 4 fractions. Fraction I, containing the doublet and 80 000, 60 000 and 46 000 subunit molecular weight bands was 5-fold purified with respect to Ca2+-ATPase activity, but was devoid of actin-activated Mg2+-ATPase activity. Fraction II, containing only the doublet, was devoid of Ca2+ and actin-activated Mg2+-ATPase activity. The 210 000 subunit molecular weight protein could be phosphorylated in the presence of Mg2+ in the crude extract and Fraction I but not in Fraction II.
采用低离子强度提取法从人红细胞膜中分离出一种具有哇巴因不敏感Ca2 + -ATP酶活性的纤维状蛋白复合物。加入兔骨骼肌肌动蛋白后可显示出Mg2 + -ATP酶活性，从而证明红细胞膜粗提物中存在类肌球蛋白蛋白。在十二烷基硫酸钠凝胶电泳中，提取物主要显示分子量为230 000和210 000的亚基条带双峰，以及10多条迁移速度更快的条带。红细胞膜提取物在Sepharose 4B上进行凝胶过滤得到4个组分。组分I含有双峰以及分子量为80 000、60 000和46 000的亚基条带，其Ca2 + -ATP酶活性纯化了5倍，但缺乏肌动蛋白激活的Mg2 + -ATP酶活性。组分II仅含有双峰，缺乏Ca2 +和肌动蛋白激活的Mg2 + -ATP酶活性。分子量为210 000的亚基蛋白在粗提物和组分I中存在Mg2 +时可被磷酸化，但在组分II中不能被磷酸化。
