Davies A, Meeran K, Cairns M T, Baldwin S A
J Biol Chem. 1987 Jul 5;262(19):9347-52.
Antibodies were raised in rabbits against synthetic peptides corresponding to the N-terminal (residues 1-15) and the C-terminal (residues 477-492) regions of the human erythrocyte glucose transporter. The antisera recognized the intact transporter in enzyme-linked immunosorbent assays (ELISA) and Western blots. In addition, the anti-C-terminal peptide antibodies were demonstrated, by competitive ELISA and by immunoadsorption experiments, to bind to the native transporter. Competitive ELISA, using intact erythrocytes, unsealed erythrocyte membranes, or membrane vesicles of known sidedness as competing antigen, showed that these antibodies bound only to the cytoplasmic surface of the membrane, indicating that the C terminus of the protein is exposed to the cytoplasm. On Western blots, the anti-N-terminal peptide antiserum labeled the glycosylated tryptic fragment of the transporter, of apparent Mr = 23,000-42,000, showing that this originates from the N-terminal half of the protein. The anti-C-terminal peptide antiserum labeled higher Mr precursors of the Mr = 18,000 tryptic fragment, although not the fragment itself, indicating that the latter, with its associated cytochalasin B binding site, is derived from the C-terminal half of the protein. Antiserum against the intact transporter recognized the C-terminal peptide on ELISA, and the Mr = 18,000 fragment but not the glycosylated tryptic fragment on Western blots.
针对人红细胞葡萄糖转运蛋白N端（第1 - 15位氨基酸残基）和C端（第477 - 492位氨基酸残基）区域的合成肽，在兔体内制备了抗体。在酶联免疫吸附测定（ELISA）和蛋白质印迹法中，这些抗血清识别完整的转运蛋白。此外，通过竞争ELISA和免疫吸附实验证明，抗C端肽抗体可与天然转运蛋白结合。使用完整红细胞、未封闭的红细胞膜或已知取向的膜泡作为竞争抗原的竞争ELISA表明，这些抗体仅与膜的细胞质表面结合，这表明该蛋白的C端暴露于细胞质中。在蛋白质印迹法中，抗N端肽抗血清标记了转运蛋白的糖基化胰蛋白酶片段，其表观分子量为23,000 - 42,000，表明该片段源自蛋白的N端一半区域。抗C端肽抗血清标记了分子量为18,000的胰蛋白酶片段的较高分子量前体，尽管未标记该片段本身，这表明后者及其相关的细胞松弛素B结合位点源自蛋白的C端一半区域。针对完整转运蛋白的抗血清在ELISA中识别C端肽，在蛋白质印迹法中识别分子量为18,000的片段，但不识别糖基化胰蛋白酶片段。
