Establishment of Peptide-Based Radioimmunoassay For Glucose Transporter Isoforms

1990 Fiscal Year Final Research Report Summary

• Project/Area Number: 63870046
• Research Category: Grant-in-Aid for Developmental Scientific Research (B).
• Allocation Type: Single-year Grants
• Research Field: 内分泌・代謝学
• Research Institution: University of Tokyo
• Principal Investigator: OKA Yoshitomo Faculty of Medicine, University of Tokyo Assistant Professor, 医学部(病), 助手 (70175256)
• Co-Investigator(Kenkyū-buntansha): AKANUMA Yasuo Institute for Diabetes Care and Research, Asahi Life Foundation Director, 所長 ; ASANO Tomoichiro Faculty of Medicine, University of Tokyo Research Associate, 医学部(病), 医員 ; SHIBASAKI Yoshikazu Faculty of Medicine, University of Tokyo Assistant Professor, 医学部(病), 助手 (80196419)
• Project Period (FY): 1988 – 1990
• Keywords: Radioimmunoassay / Glucose transporter / Synthetic Peptides

Research Abstract

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.

Research Products

• [Publications] Asano,T.,Shibasaki,Y.,Kasuga,M.,Kanazawa,Y.,Takaku,F.,Akanuma,Y.Oka,Y: "Cloning of a rabbit brain glucose transporter cDNA and alteration of glucose transporter mRNA during tissue development." Biochem Biophys Res Commun. 154ー3. 1204-1211 (1988)
  • Description: 「研究成果報告書概要(和文)」より
• [Publications] Asano,T.,Shibasaki,Y.,Ohno,S.,Taira,H.,Lin,J.,Kasuga,M.,Kanazawa,Y.,eto: "Rabbit brain glucose transporter responds to insulin when experssed in insulinーsensitive CHO cell." J Biol Chem. 264. 3416-3420 (1989)
  • Description: 「研究成果報告書概要(和文)」より
• [Publications] Asano,T.,Shibasaki,Y.,Lin,JーL,Akanuma,Y.,Takaku,F.and Oka,Y.: "The nucleotide sequence of cDNA for a mouse liverーtype glucosse transporter protein" Nucleic Acid Res.17. 6386 (1989)
  • Description: 「研究成果報告書概要(和文)」より
• [Publications] Oka,Y.,Asano,T.,Shibasaki,Y.Lin,JーL.,Tsukuda,K.,Katagiri,H.,Akanuma,Y.and TakaKu,F:"Cーteriminal truncated glucose transporter is locked into an inwardーfacing from without transport activity." Nature. 345. 550-553 (1990)
  • Description: 「研究成果報告書概要(和文)」より
• [Publications] Tsukuda,K.,Asano,T.,Lin,JーL.Katagiri,H.,Ishihara,H.,Takaku,F.and Oka,Y.: "Peptideーbased radiommunoassay specific for GLUT1 glucose transporter." Diabetes. (1991)
  • Description: 「研究成果報告書概要(和文)」より
• [Publications] Asano, T., Shibasaki, Y., Kasuga, M., Kanazawa, Y., Takaku, F., Akanuma, Y., and Oka, Y.: "Cloning of a rabbit brain glucose transporter cDNA and alteration of glucose transporter mRNA during tissue development." Biochem Biophys Res Commun. 1543. 1204-1211 (1988)
  • Description: 「研究成果報告書概要(欧文)」より
• [Publications] Asano, T., Shibasaki, Y., Ohno, S., Taira, H., Lin, J., Kasuga, M., Kanazawa, Y., Akanuma, Y., Takaku, F., and Oka, Y.: "Rabbit brain glucose transporter responds to insulin when expressed in insulin-sensitive CHO cell." J Biol Chem. 264. 3416-3420 (1989)
  • Description: 「研究成果報告書概要(欧文)」より
• [Publications] Asano, T., Shibasaki, Y., Lin, J-L, Akanuma, Y., Takaku, F. and Oka, Y.: "The nucleotide sequence of cDNA for a mouse liver-type glucosse transporter protein." Nucleic Acid Res.17. 6386 (1989)
  • Description: 「研究成果報告書概要(欧文)」より
• [Publications] Oka, Y., Asano, T., Shibasaki, Y., Lin, J-L., Tsukuda, K., Katagiri, H., Akanuma, Y. and Takaku, F.: "C-terminal truncated glucose transporter is locked into an inward-facing form without transport activity." Nature. 345. 550-553 (1990)
  • Description: 「研究成果報告書概要(欧文)」より
• [Publications] Tsukuda, K., Asano, T., Lin, J-L., Katagiri, H., Ishihara, H., Takaku, F. and Oka, Y.: "Peptide-based radioimmunoassay specific for GLUT1 glucose transporter." Diabetes. (1991)
  • Description: 「研究成果報告書概要(欧文)」より