CONFORMATIONAL CHANGE OF RABBIT AMINOPEPTIDASE-N INTO ENTEROCYTE PLASMA-MEMBRANE DOMAINS ANALYZED BY FLOW-CYTOMETRY FLUORESCENCE ENERGY-TRANSFER
CONFORMATIONAL CHANGE OF RABBIT AMINOPEPTIDASE-N INTO ENTEROCYTE PLASMA-MEMBRANE DOMAINS ANALYZED BY FLOW-CYTOMETRY FLUORESCENCE ENERGY-TRANSFER
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DOI：
10.1083/jcb.108.6.2193
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发表时间：
1989-06-01
影响因子：
7.8
通讯作者：
MAROUX, S
中科院分区：
文献类型：
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作者：
GORVEL, JP;MISHAL, Z;MAROUX, S
膜泡制剂是研究整合到极化细胞的专门质膜结构域的糖蛋白的拓扑结构的非常合适的材料。在这里，我们表明，荧光能量转移的流式细胞仪测量先前用于研究分离的细胞的表面成分之间的关系，可以应用于膜囊泡。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囊泡中的酶的寡聚化和构象状态。在猪酶整合到这两种类型的囊泡的情况下，并在兔酶整合到基底外侧膜囊泡的情况下，观察到的高荧光能量转移同意非常好的二聚体组织的存在，这是直接证明了交联实验。虽然与后一种技术，我们观察到，兔氨肽酶也二聚化刷状缘膜，没有检测到相应的囊泡的能量转移。这表明两个相关单体的相对位置不同，这取决于兔氨肽酶是瞬时整合到基底外侧膜中还是永久整合到刷状缘膜中。氨基肽酶溶解洗涤剂和胰蛋白酶释放的胞外结构域的交联表明，只有锚结构域之间的相互作用保持兔酶的二聚体结构，而ecotodomains之间的相互作用也存在于猪酶。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域的组织中的显著变化在猪酶的情况下没有发生。
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 crosslinking 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 ecotodomains 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.