Demant E J, Christiansen K, Tranum-Jensen J
Department of Biochemistry, Panum Institute, University of Copenhagen, Denmark.
Biosci Rep. 1992 Dec;12(6):471-82. doi: 10.1007/BF01122035.
Human transferrin receptor was isolated from Triton X-100 solubilized placental plasma membranes by a rapid one-step chromatographic procedure based on immunoadsorption of the receptor-transferrin complex on anti-transferrin Sepharose and lectin-affinity on wheat germ agglutinin. Following exchange of Triton X-100 with CHAPS or n-octylglucoside, the purified receptor was incorporated into egg phosphatidylcholine liposomes upon detergent removal by dialysis (lipid/protein ratio 15:1 to 45:1 (w/w)). Reconstitution of the receptor was confirmed by trypsin cleavage to dissociate the large extracellular receptor domain from the liposomal membranes. Electron micrographs of the receptor-lipid recombinants negatively stained with sodium sillicotungstate, showed that the receptor molecules distributed very inhomogeneously on the liposomes, most receptors being clustered. Single copies of the receptor were seen as elongate structures (5 x 10 nm) oriented with their long axis parallel to the liposome surface and separated from this by a 2-3 nm gap. This result provides evidence for a narrow connecting link between the globular extracellular receptor domain and the membrane spanning segment.
通过一种快速的一步色谱法从经Triton X - 100溶解的胎盘质膜中分离出人类转铁蛋白受体，该方法基于受体 - 转铁蛋白复合物在抗转铁蛋白琼脂糖上的免疫吸附以及在麦胚凝集素上的凝集素亲和作用。在用CHAPS或正辛基葡糖苷替换Triton X - 100后，通过透析去除去污剂，将纯化的受体掺入到卵磷脂酰胆碱脂质体中（脂质/蛋白质比例为15:1至45:1（w/w））。通过胰蛋白酶切割以从脂质体膜上解离大的细胞外受体结构域来确认受体的重构。用硅钨酸钠负染的受体 - 脂质重组体的电子显微镜照片显示，受体分子在脂质体上分布非常不均匀，大多数受体聚集在一起。单个受体拷贝呈现为细长结构（5×10纳米），其长轴与脂质体表面平行，并与之相隔2 - 3纳米的间隙。这一结果为球状细胞外受体结构域与跨膜区段之间存在狭窄的连接提供了证据。
