| Project/Area Number |
03557033
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| Research Category |
Grant-in-Aid for Developmental Scientific Research (B)
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| Allocation Type | Single-year Grants |
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| Research Field |
内科学一般
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| Research Institution | Osaka University |
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Principal Investigator |
AWAMORI Ryuzo First Dept. Med. Osaka University, 医学部・第一内科, 講師 (00116021)
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| Co-Investigator(Kenkyū-buntansha) |
WATARAI Takao Osaka Univ. Hospital Senior Resident, 医学部・附属病院第一内科, 助手
IWAMA Norimichi Osaka Univ. Hospital Senior Resident, 医学部・附属病院第一内科, 助手
MORISHIMA Toyohiko Osaka Univ. Medicine Assistant Teacher, 医学部・第一内科, 助手 (50221635)
YAMAZAKI Yoshimitsu Osaka Univ. Medicine Assistant Teacher, 医学部・第一内科, 助手 (40201834)
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| Project Period (FY) |
1991 – 1992
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| Project Status |
Completed (Fiscal Year 1992)
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| Budget Amount *help |
¥5,400,000 (Direct Cost: ¥5,400,000)
Fiscal Year 1992: ¥1,100,000 (Direct Cost: ¥1,100,000)
Fiscal Year 1991: ¥4,300,000 (Direct Cost: ¥4,300,000)
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| Keywords | Insulin biosensor / insulin-EGF chimeric receptor / artophosphorylation / インスリン / EGF / キメラ / 燐酸化 |
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| Research Abstract |
Cloned human insulin receptor and EGF receptor complementary DNAs were jointed and used in combination with SV40 early promoter transcriptional elements to generate expression construct cording for complete chimeric receptor. This chimeric receptor DNA was inserted into the plasmid pUC12. This plasmid was transfected into COS-7 cells by the calcium phosphate precipitation method. After 48 hr of incubation, insulin-EGF chimeric receptor was partially purified by using WGA-agarose column. We investigated whether this insulin-EGF chimeric receptor was autophosphorylated in a dose-dependent manner.
Analysis by SDS-polyacrylamide gel electrophoresis revealed ^<32>P incorporation into 120KDa and 95KDa proteins, suggesting that 95KDa protein was intrinsic beta subunit of insulin receptor in COS-7 cell and 120KDa protein was beta subunit of transfected insulin-EGF chimeric receptor. Although both insulin receptor and insulin-EGF chimeric receptor were phosphorylated in a dose- dependent manner(insulin concentration, 0-77nM), insulin-EGF chimeric receptor was more highly autophosphorylated than insulin receptor.
These results indicate that this insulin-EGF chimeric receptor can be useful as an insulin biosensor.
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