PURIFICATION AND CRYSTALLIZATION OF THE CALCIUM-BINDING PROTEIN OF SARCOPLASMIC-RETICULUM FROM SKELETAL-MUSCLE
PURIFICATION AND CRYSTALLIZATION OF THE CALCIUM-BINDING PROTEIN OF SARCOPLASMIC-RETICULUM FROM SKELETAL-MUSCLE
复制标题
DOI：
10.1073/pnas.82.12.4036
复制
发表时间：
1985-01-01
影响因子：
11.1
通讯作者：
FLEISCHER, S
中科院分区：
文献类型：
--
作者：
MAURER, A;TANAKA, M;FLEISCHER, S
[兔]骨骼肌肌浆网的Ca结合蛋白（也称为钙螯合蛋白）通过从具有去污剂八甘醇单正十二烷基醚（C12 E8）的囊泡的隔室释放并通过随后用特定的二价阳离子沉淀Ca结合蛋白来纯化。分离的蛋白质在NaDodSO 4/PAGE [十二烷基硫酸钠/聚丙烯酰胺凝胶电泳]上显示单一条带，并且每mg蛋白质结合903 nmol的Ca 2+。Ca ~（2+）、Mg ~（2+）、Sr ~（2+）或这3种阳离子的组合在较窄的浓度范围内均可使Ca ~（2+）结合蛋白结晶。最高达500倍的针状晶体。得到50 μ m。二价阳离子的去除导致晶体的溶解。晶体的间距和角度通过EM使用3种不同的样品制备方法获得。通过冷冻干燥和负染色EM，确定沿着轴a和B的间距各为10-11 nm，2个轴之间的角度为90 °。通过薄切片EM，沿沿着轴a的间隔为11 nm，沿沿着轴c的间隔为15-16 nm，并且两个轴之间的角度为75 °。本研究报告了一种简单而快速的方法用于纯化的Ca结合蛋白;条件结晶的蛋白质使用Ca 2+，Mg 2+，Sr 2+，或3的组合;和一些初步的晶体特性。通过EM和X射线衍射表征了晶体性质。较大的晶体衍射超过3埃。布拉格间距。
The Ca binding protein of [rabbit] skeletal muscle sarcoplasmic reticulum (also referred to as calsequestrin) was purified by release from the compartment of the vesicles with the detergent octaethyleneglycol mono-n-dodecyl ether (C12E8) and by subsequent precipitation of the Ca binding protein with specific divalent cations. The isolated protein exhibited a single band on NaDodSO4/PAGE [sodium dodecyl sulfate/polyacrylamide gel electrophoresis] and bound 903 nmol of Ca2+ per mg of protein. The Ca binding protein could be crystallized in the presence of Ca2+, Mg2+, Sr2+, or combinations of these 3 cations used in a narrow concentration range. Needle-shaped crystals of up to 500 .times. 50 .mu.m were obtained. The removal of the divalent cations resulted in solubilization of the crystals. The spacings and angles of the crystals were obtained by EM using 3 different methods of sample preparation. By freeze-drying and negative staining EM, the spacings along axes a and b were determined to be 10-11 nm each, and the angle between the 2 axes was 90.degree.. By thin section EM, the spacing along axis a was 11 nm, along axis c was 15-16 nm, and the angle between the 2 axes was 75.degree.. This study reports a simple and rapid method for purification of the Ca binding protein; conditions to crystallize the protein using Ca2+, Mg2+, Sr2+, or combinations of the 3; and some preliminary characteristics of the crystals. The crystalline nature was characterized by EM and X-ray diffraction. The larger crystals diffracted beyond 3-.ANG. Bragg spacing.