Lüdi H, Hasselbach W
Biochim Biophys Acta. 1985 Nov 21;821(1):137-41. doi: 10.1016/0005-2736(85)90164-6.
Sarcoplasmic reticulum vesicles from fast skeletal muscle were partially delipidated with sodium cholate at high ionic strength and sedimented in a discontinuous sucrose gradient. Phospholipid content was reduced from 0.777 mumol/mg protein to 0.242 mumol/mg protein. As judged from gel electrophoresis and high pressure liquid gel chromatography, accessory proteins were removed during centrifugation and the Ca2+-ATPase was obtained in an almost pure form. Addition of myristoylglycerophosphocholine (1 mg/mg protein) reactivates ATPase and dinitrophenylphosphatase activity to the same degree obtained with native vesicles. Using the analytical ultracentrifuge it could be demonstrated that the reactivated Ca2+-ATPase was present exclusively in a monomeric state. These results were obtained at high and low ionic strength and up to a protein concentration of 10 mg/ml. Therefore this preparation should be very useful to investigate differences between oligomeric and monomeric Ca2+-ATPase.
来自快速骨骼肌的肌浆网囊泡在高离子强度下用胆酸钠进行部分脱脂，并在不连续蔗糖梯度中沉降。磷脂含量从0.777微摩尔/毫克蛋白质降至0.242微摩尔/毫克蛋白质。从凝胶电泳和高压液相凝胶色谱判断，辅助蛋白在离心过程中被去除，并且以几乎纯的形式获得了Ca2 + -ATP酶。添加肉豆蔻酰甘油磷酸胆碱（1毫克/毫克蛋白质）可将ATP酶和二硝基苯磷酸酶活性重新激活至与天然囊泡相同的程度。使用分析超速离心机可以证明，重新激活的Ca2 + -ATP酶仅以单体状态存在。这些结果是在高离子强度和低离子强度下以及高达10毫克/毫升的蛋白质浓度下获得的。因此，该制剂对于研究寡聚体和单体Ca2 + -ATP酶之间的差异应该非常有用。
