Characterization of reaction centers from photosynthetic bacteria. II. Amino acid composition of the reaction center protein and its subunits in Rhodopseudomonas spheroides R-26.
Characterization of reaction centers from photosynthetic bacteria. II. Amino acid composition of the reaction center protein and its subunits in Rhodopseudomonas spheroides R-26.
复制标题
光合细菌反应中心的表征。
DOI：
10.1021/bi00704a014
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发表时间：
1974
期刊：
影响因子：
2.9
通讯作者：
G. Feher
中科院分区：
文献类型：
--
作者：
L. Steiner;M. Okamura;A. D. Lopes;E. Moskowitz;G. Feher
材料与方法反应中心的制备。反应中心的制备。spheroides R-26和R. RubrumG-9描述于随附的论文（Okamura等人，1974年）。含有放射性标记的半胱氨酸或色氨酸的反应中心从在不含酵母提取物、蛋白胨或谷氨酸盐的改良Hutner培养基中生长的细菌中获得，但包括100 gCi的L-[3sS]胱氨酸/l。（施瓦茨/曼，sp act. 25 mCi/mmol）加200 mg L-甲硫氨酸/l。或200 pd dl-[14 C]色氨酸/l。（International Chemicaland Nuclear Corp.，环-2标记，分离。3 mCi/mmol）。在每种情况下，包括浓度为10 mg/l的其他氨基酸。在十二烷基硫酸钠中的稀释性聚丙烯酰胺凝胶电泳。制备型凝胶电泳的程序基本上与分析型凝胶电泳的程序相同，在前面的论文中讨论过，不同的是每个凝胶的负载量约为50 pg。电泳后，用参考刚毛标记凝胶，并在280 nm处扫描。用拉紧线和模板从20个凝胶上切下对应于蛋白条带的切片，并在等于凝胶切片体积的1.5倍体积的缓冲液（50 mM Tris-Cl（pH 8.0）-0.1%十二烷基硫酸钠）中浸渍。然后将浸软的凝胶切片置于电泳管（12 X 0.8 cm）中，电泳管底部用尼龙网支撑的0.5 cm长的10%聚丙烯酰胺塞密封。将试管置于同心圆筒（2x2 cm）中，圆筒一端用透析膜密封，并含有相同的缓冲溶液。将蛋白质通过毛细管洗脱到这些柱中（15 mA/凝胶，4小时），并通过真空透析在火棉胶袋（Schleicherand Schuell）中浓缩。分离的纯度
Materials and MethodsPreparation of Reaction Centers. The preparation of re-action centers from R. spheroides R-26 and R. rubrumG-9 is described in the accompanying paper (Okamura et al., 1974). Reaction centers containing either radiolabeled half-cystine or tryptophan were obtained from bacteria grown in modified Hutner medium without yeast extract, peptone, or glutamate, but including either 100 gCi of L-[3sS] cystine/l.(Schwarz/Mann, sp act. 25 mCi/mmol) plus 200 mg of L-methionine/1. or 200 pd of dl-[14C] tryptophan/l.(International Chemicaland Nuclear Corp., ring-2 labeled, sp act. 3 mCi/mmol). In each case, the other amino acids were included at a concentration of 10mg/l. Preparative Polyacrylamide Gel Electrophoresis in Sodium Dodecyl Sulfate. The procedure for preparative gel electrophoresis was essentially the same as that for analytical gel electrophoresis, discussed in the preceding paper, except that the load per gel was about 50 pg. After electrophoresis the gels were marked with a reference bristle and scanned at 280 nm. Sections corresponding to the protein bands were excised from 20 gels with a taut wire and template and macerated in a volume of buffer (50 mM Tris-Cl (pH 8.0)-0.1% sodium dodecyl sulfate) equal to 1.5 times the volume of the gel slices. The macerated gel sections were then placed into electrophoresis tubes (12 X 0.8 cm) sealed at the bottom with a 0.5 cm long plug of 10% polyacrylamide supported with a nylon net. The tubes were placed into concentric cylinders (2x2 cm) that were sealed at one end with a dialysis mem-brane and that containedthe same buffer solution. The protein was eluted electrophoretically into these cylinders (15 mA/gel for 4 hr) and concentrated by vacuum dialysis in collodion bags (Schleicherand Schuell). The purity of the separated