Structure and Mechanism of Signal Transduction of GHRH Receptor,and its Clinical Significance.
Grant-in-Aid for General Scientific Research (B)
|Allocation Type||Single-year Grants|
|Research Institution||FIRST DEPARTMENT OF INTERNAL MEDICINE,SCHOOL OF MEDICINE,THE UNIVERSITY OF TOKUSHIMA|
SAITO Shiro SCHOOL OF MEDICINE,UNIVERSITY OF TOKUSHIMA,PROFESSOR, 医学部, 教授 (40010368)
YOKOGOSHI Yutaka SCHOOL OF MEDICINE,UNIVERSITY OF TOKUSHIMA,INSTRUCTOR, 医学部, 助手 (00253203)
SHINTANI Yasumi UNIVERSITY OF TOKUSHIMA,HOSPITAL,INSTRUCTOR, 助手 (10235773)
吉本 勝彦 徳島大学, 医学部附属病院, 助手 (90201863)
山崎 柳一 徳島大学, 医学部附属病院, 助手 (10191256)
|Project Period (FY)
1990 – 1991
Completed(Fiscal Year 1991)
|Budget Amount *help
¥6,400,000 (Direct Cost : ¥6,400,000)
Fiscal Year 1991 : ¥800,000 (Direct Cost : ¥800,000)
Fiscal Year 1990 : ¥5,600,000 (Direct Cost : ¥5,600,000)
|Keywords||GHRH receptor / Plasma GHRH binding proteins / GHRH agonist / Molecular weight / Isoelectric point / レセプタ-アッセイ / クロスリンク|
We identified growth hormone-releasing hormone(GHRH) binding activity from human placental membrane and characterized it as follows.
1) Identification of GHRH binding activity
GHRH binding activity was identified using [D-Ala^2,Ala^<15>,Nle^<27>,Lys^<30> (Biot)]hGHRH (1-30)NH_2 as a ligand from pig pituitary and human placental membrane fractions. The biologically active binding proteins were solubilized from placental membrane using 10mM CHAPS. Gel filtration using Sephacryl-S300 column showed 3 peaks of the binding activities of 669Kd, 67Kd, and 45-17Kd.
2) Purification and characterization of a GHRH binding protein
A GHRH binding protein solubilized from human placental membrane was purified to a single protein band on silver staining using [D-Ala^2,Ala^<15>,Nle^<27>,Lys^<30>(Biot)]hGHRH (1-30)NH_2-immobilized column, GS3000SW, and reverse phase HPLC. The purified protein showed a protein band of 75Kd on SDS-PAGE and its isoelectric point was pH 4.5-5.0.
3) PCR-cloning of GHRH binding proteins
Depending on the information of reported cDNA sequence of GHRH receptor, PCR-cloning of genomic DNA of GHRH receptor was performed. PCR product showed multiple bands on agarose-gel electrophoresis, suggesting the presence of the subtype of GHRH receptor or related proteins.
4) Identification of plasma GHRH binding proteins
Human plasma protein binded specifically with GHRH. The gel filtration of the cross-linked proteins showed four peaks of GHRH binding activities of 669kd, 200-100Kd, 67kd, and 45-17Kd.
The purification and protein sequencing analysis of other molecular form of GHRH binding proteins are in progress.
Research Products (12results)