Francis G A, Richards W R
Biochemistry. 1980 Oct 28;19(22):5104-11. doi: 10.1021/bi00563a026.
Reduction with [3H]KBH4 of Schiff's bases generated by reaction with pyridoxal 5'-phosphate (which cannot penetrate the intact cytoplasmic membrane) yields tritium-labeled derivatives of both proteins and lipids accessible on the periplasmic side of the cytoplasmic membrane. Application of this technique to phototrophically grown Rhodopseudomonas sphaeroides labeled both the cell envelope and chromatophore fractions. The technique was also applied to R. sphaeroides harvested at various times during an adaptation from heterotrophic to phototrophic growth conditions. The specific activity of the chromatophore fraction after 20 h of adaptation was 76% of that found at the beginning, indicating that the intracytoplasmic membranes and cytoplasmic membrane form a continuous membrane system, with the majority of the intracytoplasmic membranes accessible to the external medium throughout the adaptation. The identity of the proteins labeled by this technique was investigated in two fractions labeled after cell disruption: normal "inside-out" chromatophores and "right-side-out" membrane vesicles isolated by lysozyme--osmotic shock treatment of cells grown in high light intensity (15000 lx). The results after sodium dodecyl sulfate--polyacrylamide gel electrophoresis and fluorography indicated that the 28000-dalton subunit (and to a lesser extent the 21000-dalton subunit) of the reaction center complex and two polypeptides in the light-harvesting region of the gel were heavily labeled in the chromatophores and were thus accessible on the cytoplasmic side of the membrane. At least one of the latter two polypeptides was also labeled in the membrane vesicles and was thus also accessible on the periplasmic side of the membrane. None of the reaction center subunits was significantly labeled in a reaction center complex prepared from the membrane vesicle sample.
用[³H]KBH₄还原与磷酸吡哆醛（无法穿透完整的细胞质膜）反应生成的席夫碱，可得到细胞质膜周质侧可及的蛋白质和脂质的氚标记衍生物。将该技术应用于光合生长的球形红假单胞菌，标记了细胞包膜和类囊体组分。该技术还应用于在从异养生长条件适应光合生长条件的不同时间收获的球形红假单胞菌。适应20小时后类囊体组分的比活性为开始时的76%，表明细胞质内膜和细胞质膜形成一个连续的膜系统，在整个适应过程中大部分细胞质内膜可与外部介质接触。通过该技术标记的蛋白质的身份在细胞破碎后标记的两个组分中进行了研究：正常的“内翻”类囊体和通过对高光强（15000勒克斯）下生长的细胞进行溶菌酶 - 渗透压休克处理分离的“外翻”膜泡。十二烷基硫酸钠 - 聚丙烯酰胺凝胶电泳和荧光自显影的结果表明，反应中心复合物的28000道尔顿亚基（以及程度较轻的21000道尔顿亚基）和凝胶光捕获区域的两种多肽在类囊体中被大量标记，因此在膜的细胞质侧是可及的。后两种多肽中至少有一种在膜泡中也被标记，因此在膜的周质侧也是可及的。在从膜泡样品制备的反应中心复合物中，没有一个反应中心亚基被显著标记。
