Secondary structure and assembly mechanism of an oligomeric channel protein.
Secondary structure and assembly mechanism of an oligomeric channel protein.
复制标题
寡聚通道蛋白的二级结构和组装机制。
DOI：
10.1021/bi00329a017
复制
发表时间：
1985
期刊：
影响因子：
2.9
通讯作者：
Bayley,H
中科院分区：
文献类型：
--
作者：
Tobkes,N;Wallace,BA;Bayley,H
材料与方法金黄色葡萄球菌（木株）来自美国活字培养收藏。用Schleicher & Schuell公司的胶袋（UH100/10）进行真空透析。从Sigma提取的去氧胆酸（DOC） 1用乙醇/水重结晶。热溶液首先用木炭处理。其他材料如下：电核公司的受控多孔玻璃微珠（CPG 00350）， machery - nagel公司的纤维素薄层色谱板，Pierce公司的亚酰咪啶二甲酯，Boehringer-Mannheim公司的蛋白酶K， Sigma公司的tpck处理胰蛋白酶，Sigma公司的苯甲磺酰氟，以及Amersham International公司的Na125I。a-毒素的制备从金黄色葡萄球菌培养物的上清液中分离a-毒素的方法与Cassidy & Harshman（1976）的方法非常相似。在控制孔玻璃珠上吸附层析后，用超滤浓缩，在Sephadex G-50柱上进一步分离。纯化后的毒素经10 mM Tris-HCl-150 mM NaCl， pH 8.0真空透析浓缩至~ 2 mg/mL，在70℃下保存。
Materials and Methods Materials. S. aureus (Wood strain) was obtained from the American Type Culture Collection. Vacuum dialysis was carried out with collodion bags (UH100/10) from Schleicher & Schuell. Deoxycholic acid (DOC) 1 from Sigma was re-crystallized from ethanol/water. The hot solution was first treated with charcoal. Other materials were obtained as follows: controlled poreglass beads (CPG 00350) from Electro-Nucleonics, cellulose thin-layer chromatography plates from Macherey-Nagel, dimethyl suberimidate from Pierce, proteinase K from Boehringer-Mannheim, TPCK-treated trypsin from Sigma, phenylmethanesulfonyl fluoride from Sigma, and Na125I from Amersham International. Preparation of a-Toxin. a-Toxin was isolated from the supernatants of cultures of S. aureus by a method closely similar to that of Cassidy & Harshman (1976). All steps were at 4 C. After adsorption chromatography on controlled pore glass beads, the toxin-containing fractions were concentrated by ultrafiltration and further fractionated on a Sephadex G-50 column. The purified toxin was concentrated to~ 2 mg/mL by vacuum dialysis against 10 mM Tris-HCl-150 mM NaCl, pH 8.0, and stored at-70 C.