Establishment of Peptide-Based Radioimmunoassay For Glucose Transporter Isoforms

通过使用基于GLUT1 cDNA序列的合成肽，开发了GLUT1葡萄糖转运蛋白的放射免疫测定法（RIA）。合成对应于GLUT 1葡萄糖转运蛋白的羧基末端结构域的肽（ThrProGluGluLeuPheHisProLeuGlyAlaAspSerGlnVal），并通过肽的氨基末端与钥孔血蓝蛋白缀合。针对该复合物产生抗体，并使用固定化肽进行亲和纯化。合成了在上述肽的氨基末端添加有酪氨酸残基的第二肽，并用作标准品，还碘化用于制备放射性配体。该测定法对GLUT 1葡萄糖转运蛋白的羧基末端结构域具有高度重现性、灵敏度和特异性。与其他葡萄糖转运蛋白亚型GLUT 2和GLUT 4无交叉反应性。此外，本RIA对人红细胞中葡萄糖转运蛋白数量的测定结果与以前基于细胞松弛素B结合的报道相当一致，表明本RIA能够定量葡萄糖转运蛋白的数量。该测定在4小时内完成，可用于同时测量许多样品中的GLUT1。此外，它还可用于多种组织中GLUT 1的测定。

A radioimmunoassay (RIA) for the GLUT1 glucose transporter has been developed by using a synthesized peptide based upon the sequence of the cDNA for GLUTI1. A peptide corresponding to the carboxy-terminal domain of the GLUT1 glucose transporter (ThrProGluGluLeuPheHisProLeuGlyAlaAspSerGlnVal) was synthesized and conjugated to keyhole limpet hemocyanin through the amino-terminus of the peptide. An antibody was raised against this complex and affinity-purified using the immobilized peptide. A second peptide with tyrosine residue added to amino-terminus of the above peptide was synthesized and used as a standard and also iodinated for preparation of the radioactive ligand. The assay is highly reproducible, sensitive, and specific for the carboxy-terminal domain of the GLUT1 glucose transporter. It has no cross-reactivity with the other glucose transporter isoforms, GLUT2 and GLUT4. Furthermore, the results obtained with this RIA on the number of glucose transporters in human erythrocytes are in a fairly good agreement with the previous reports based on cytochalasin B binding, suggesting that this RIA is able to quantify the number of glucose transporters. The assay is completed within 4h and can be used for simultaneous measurement of GLUT1 in many samples. In addition, it can be applied to the measurement of GLUT1 in several types of tissue.