STRUCTURAL STUDIES ON THE MEMBRANE-BOUND IMMUNOGLOBULIN E-RECEPTOR COMPLEX .2. MAPPING OF DISTANCES BETWEEN SITES ON IGE AND THE MEMBRANE-SURFACE
STRUCTURAL STUDIES ON THE MEMBRANE-BOUND IMMUNOGLOBULIN E-RECEPTOR COMPLEX .2. MAPPING OF DISTANCES BETWEEN SITES ON IGE AND THE MEMBRANE-SURFACE
复制标题
DOI：
10.1021/bi00283a026
复制
发表时间：
1983-01-01
期刊：
影响因子：
2.9
通讯作者：
BAIRD, B
中科院分区：
文献类型：
--
作者：
HOLOWKA, D;BAIRD, B
Resonance energy transfer was used to investigate the structure of IgE bound to receptors on the plasma membrane of rat basophilic leukemia (RBL) cells. Isolated monoclonal IgE was labeled with donor probes in 2 different regions: fluorescein 5-isothiocyanate preferentially labels the Fab segments, and limited reduction followed by alkylation with N-[4-[7-(diethylamino)-4-methylcoumarin-3-yl]phenyl]maleimide causes selective modification of the inter .epsilon. chain disulfides in the C.epsilon.2 domains [2nd domain of the IgE constant region]. These donor-labeled IgE molecules bind with characteristic high affinity to large, oriented plasma membrane vesicles prepared from RBL cells, and amphipathic acceptor probes can be titrated into these vesicles at surface densities that can be directly measured. Two different acceptor probes for each donor were used. From fluorescence quenching of the donor probes measured as a function of acceptor density, the effective distance from the membrane surface was determined to be 71-106 .ANG. for the fluorescein-labeled Fab segments and 35-52 .ANG. for the coumarin-labeled C.epsilon.2 domains. This information is used together with that from recent biochemical studies to propose new models for the structural orientation of membrane receptor-bound IgE.
