Max-Planck-Institut für Biochemie, D-8033 Martinsried/München, FRG.
EMBO J. 1982;1(10):1177-83. doi: 10.1002/j.1460-2075.1982.tb00010.x.
Halorhodopsin (HR) was reconstituted in cell vesicles prepared from Halobacterium halobium strain L-07 by addition of tritium-labelled retinal and subsequently reduced with cyanoborohydride. Lysis of the labelled vesicles in water and dissolution of the cell membranes with 4% SDS allowed the purification of the retinyl protein (RP) by a 3-step procedure. Gel filtration on AcA-44 ultrogel was followed by chromatography on hydroxylapatite and preparative SDS-polyacrylamide gel electrophoresis. This procedure yielded material which migrated as a single band of an apparent mol. wt. of 25 000 on analytical SDS-polyacrylamide gels. The purification was 400-fold with an overall yield of 15%. Not only the mol. wts. but also the amino acid compositions of the RPs from bacteriorhodopsin (BR) and HR are very similar. Polyclonal antibodies against BR and HR did not, however, crossreact. When the two RPs were partially digested with staphylococcal V8 protease the proteolytic pattern of the retinyl peptides was similar, but not identical: two extra peptides are present in BR. The same kind of differences were found in the h.p.l.c. elution profiles of retinyl peptides produced by subtilisin digestion. Therefore, the two proteins must be different gene products and not modification products of one and the same protein.
盐菌紫膜蛋白（HR）通过添加氚标记的视黄醛并随后用氰基硼氢化钠还原，在从嗜盐菌 L-07 制备的囊泡中重新组成。在水中裂解标记的囊泡并用 4％SDS 溶解细胞膜，允许通过 3 步程序纯化视黄醛蛋白（RP）。在 AcA-44 超凝胶上进行凝胶过滤，然后在羟基磷灰石上进行色谱分离和制备 SDS-聚丙烯酰胺凝胶电泳。该程序产生的物质在分析 SDS-聚丙烯酰胺凝胶上迁移为表观分子量为 25000 的单一带。纯化倍数为 400 倍，总收率为 15％。不仅 RP 的分子量，而且 BR 和 HR 的氨基酸组成也非常相似。然而，针对 BR 和 HR 的多克隆抗体并未发生交叉反应。当用葡萄球菌 V8 蛋白酶部分消化这两种 RP 时，视黄醛肽的蛋白水解模式相似，但不完全相同：BR 中存在两个额外的肽。枯草杆菌蛋白酶消化产生的视黄醛肽的 h.p.l.c.洗脱曲线也存在相同的差异。因此，这两种蛋白质必须是不同的基因产物，而不是同一蛋白质的修饰产物。
