Haspel H C, Rosenfeld M G, Rosen O M
Program of Molecular Biology, Memorial Sloan-Kettering Cancer Center, New York, New York 10021.
J Biol Chem. 1988 Jan 5;263(1):398-403.
Peptides corresponding to amino acid residues 1-12 of the amino terminal and 480-492 of the carboxyl terminal of the deduced sequence of the glucose transporter were synthesized and used to produce site-specific polyclonal antipeptide sera. In a solid-phase radioimmunoassay, antiserum to the carboxyl terminal recognizes peptide 480-492 and purified human erythrocyte glucose transporter, but not peptide 1-12. Antiserum to the amino terminal recognizes peptide 1-12 but neither peptide 480-492 nor the erythrocyte transporter. The antiserum to the carboxyl terminal specifically immunoblots the Mr 55,000 glucose transporter in erythrocyte membranes and the purified erythrocyte transporter. It also recognizes a Mr 40,000-60,000 polypeptide in membranes of cells derived from different mammalian species and tissues including insulin-sensitive rat adipocytes as well as a Mr 20,000 tryptic fragment of the transporter which contains the site for photolabeling by cytochalasin B. Antiserum to the carboxyl terminal of the transporter binds specifically to leaky erythrocyte membranes but not to intact erythrocytes. This binding is saturable and competitively inhibited by peptide 480-492. Using immunofluorescence microscopy, this antiserum detects glucose transporter protein in permeabilized erythrocytes, but not in intact erythrocytes. These studies provide immunochemical evidence in support of the predicted cytoplasmic orientation of the carboxyl terminus of the glucose transporter, allow us to suggest a spatial relationship of the cytochalasin B binding site to the carboxyl terminal of the glucose transporter and suggest that antisera directed to the carboxyl terminal domain of the protein may be useful for the immunocytochemical localization of the glucose transporter.
合成了与葡萄糖转运蛋白推导序列氨基末端的1 - 12个氨基酸残基以及羧基末端的480 - 492个氨基酸残基相对应的肽段，并用于制备位点特异性多克隆抗肽血清。在固相放射免疫测定中，羧基末端抗血清可识别肽段480 - 492和纯化的人红细胞葡萄糖转运蛋白，但不能识别肽段1 - 12。氨基末端抗血清可识别肽段1 - 12，但不能识别肽段480 - 492和红细胞转运蛋白。羧基末端抗血清能特异性地对红细胞膜和纯化的红细胞转运蛋白中的55,000 Mr葡萄糖转运蛋白进行免疫印迹。它还能识别来自不同哺乳动物物种和组织（包括胰岛素敏感的大鼠脂肪细胞）的细胞膜中的一种40,000 - 60,000 Mr多肽以及该转运蛋白的一个20,000 Mr胰蛋白酶片段，该片段包含细胞松弛素B进行光标记的位点。转运蛋白羧基末端的抗血清能特异性地与渗漏的红细胞膜结合，但不与完整红细胞结合。这种结合是可饱和的，并受到肽段480 - 492的竞争性抑制。使用免疫荧光显微镜，这种抗血清可检测通透红细胞中的葡萄糖转运蛋白，但不能检测完整红细胞中的。这些研究提供了免疫化学证据，支持葡萄糖转运蛋白羧基末端预测的胞质取向，使我们能够提出细胞松弛素B结合位点与葡萄糖转运蛋白羧基末端的空间关系，并表明针对该蛋白羧基末端结构域的抗血清可能有助于葡萄糖转运蛋白的免疫细胞化学定位。
