MONOCLONAL-ANTIBODIES AGAINST THE LAC CARRIER PROTEIN FROM ESCHERICHIA-COLI .2. BINDING-STUDIES WITH MEMBRANE-VESICLES AND PROTEOLIPOSOMES RECONSTITUTED WITH PURIFIED LAC CARRIER PROTEIN
MONOCLONAL-ANTIBODIES AGAINST THE LAC CARRIER PROTEIN FROM ESCHERICHIA-COLI .2. BINDING-STUDIES WITH MEMBRANE-VESICLES AND PROTEOLIPOSOMES RECONSTITUTED WITH PURIFIED LAC CARRIER PROTEIN
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DOI：
10.1021/bi00311a018
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发表时间：
1984-01-01
期刊：
影响因子：
2.9
通讯作者：
KABACK, HR
中科院分区：
文献类型：
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作者：
HERZLINGER, D;VIITANEN, P;KABACK, HR
单克隆抗体4 b1和5 F7与乳糖载体蛋白中不同的非重叠表位结合。通过使用免疫荧光显微镜和放射性标记的单克隆抗体和Fab片段，它表明，4 B1和5 F7结合到原生质球和右侧出囊泡，但只有在很小的程度上内面出囊泡。因此，lac载体蛋白在大肠杆菌细胞质膜内具有不对称取向。大肠杆菌，并且两个表位都位于周质表面上。在右侧外囊泡中，放射性标记的4 B1以1 mol抗体/2 mol lac载体蛋白的化学计量结合，而放射性标记的4 B1 Fab片段以1：1结合。重要的是，完整的抗体及其Fab片段与用纯化的lac载体蛋白重构的蛋白脂质体结合，其化学计量与在右侧外膜囊泡中观察到的非常相似。因此，乳糖载体蛋白在重组系统中的取向可能与细菌细胞质膜中的取向相似，至少在4 B1表位方面是如此。
Monoclonal antibodies 4b1 and 5F7 bind to distinct, nonoverlapping epitopes in the lac carrier protein. By use of immunofluorescence microscopy and radiolabeled monoclonal antibodies and Fab fragments, it is shown that both 4B1 and 5F7 bind to spheroplasts and to right-side-out vesicles, but only to a small extent to inside-out vesicles. Therefore, the lac carrier protein has an asymmetric orientation within the cytoplasmic membrane of E. coli, and both epitopes are located on the periplasmic surface. In right-side-out vesicles, radiolabeled 4B1 binds with a stiochiometry of 1 mol of antibody /2 mol of lac carrier protein, while radiolabeled 4B1 Fab fragments bind 1:1. Importantly, the intact antibody and its Fab fragments bind to proteoliposomes reconstituted with purified lac carrier protein with a stoichiometry very similar to that observed in right-side-out membrane vesicles. Thus, the orientation of the lac carrier protein in the reconstituted system is probably similar to that in the bacterial cytoplasmic membrane, at least with respect to 4B1 epitope.