Gorvel J P, Mishal Z, Liegey F, Rigal A, Maroux S
Centre de Biochimie et de Biologie Moléculaire, Centre National de la Recherche Scientifique, Marseille, France.
J Cell Biol. 1989 Jun;108(6):2193-200. doi: 10.1083/jcb.108.6.2193.
Membrane vesicle preparations are very appropriate material for studying the topology of glycoproteins integrated into specialized plasma membrane domains of polarized cells. Here we show that the flow cytometric measurement of fluorescence energy transfer used previously to study the relationship between surface components of isolated cells can be applied to membrane vesicles. The fluorescein and rhodamine derivatives of a monoclonal antibody (4H7.1) that recognized one common epitope of the rabbit and pig aminopeptidase N were used for probing the oligomerization and conformational states of the enzyme integrated into the brush border and basolateral membrane vesicles prepared from rabbit and pig enterocytes. The high fluorescent energy transfer observed in the case of pig enzyme integrated into both types of vesicles and in the case of the rabbit enzyme integrated into basolateral membrane vesicles agreed very well with the existence of a dimeric organization, which was directly demonstrated by cross-linking experiments. Although with the latter technique we observed that the rabbit aminopeptidase was also dimerized in the brush border membrane, no energy transfer was detected with the corresponding vesicles. This indicates that the relative positions of two associated monomers differ depending on whether the rabbit aminopeptidase is transiently integrated into the basolateral membrane or permanently integrated into the brush border membrane. Cross-linking of aminopeptidases solubilized by detergent and of their ectodomains liberated by trypsin showed that only interactions between anchor domains maintained the dimeric structure of rabbit enzyme whereas interactions between ectodomains also exist in the pig enzyme. This might explain why the noticeable change in the organization of the two ectodomains observed in the case of rabbit aminopeptidase N does not occur in the case of pig enzyme.
膜泡制剂是研究整合到极化细胞特化质膜结构域中的糖蛋白拓扑结构的非常合适的材料。在这里我们表明，先前用于研究分离细胞表面成分之间关系的荧光能量转移的流式细胞术测量可应用于膜泡。一种识别兔和猪氨肽酶N的一个共同表位的单克隆抗体（4H7.1）的荧光素和罗丹明衍生物被用于探测整合到从兔和猪肠细胞制备的刷状缘和基底外侧膜泡中的酶的寡聚化和构象状态。在整合到两种类型膜泡中的猪酶以及整合到基底外侧膜泡中的兔酶的情况下观察到的高荧光能量转移与二聚体组织的存在非常吻合，这通过交联实验直接证明。尽管用后一种技术我们观察到兔氨肽酶在刷状缘膜中也二聚化，但相应的膜泡未检测到能量转移。这表明两个相关单体的相对位置取决于兔氨肽酶是暂时整合到基底外侧膜中还是永久整合到刷状缘膜中。用去污剂溶解的氨肽酶及其经胰蛋白酶释放的胞外结构域的交联表明，只有锚定结构域之间的相互作用维持兔酶的二聚体结构，而猪酶中也存在胞外结构域之间的相互作用。这可能解释了为什么在兔氨肽酶N的情况下观察到的两个胞外结构域组织的明显变化在猪酶的情况下没有发生。
