| Project/Area Number |
63571071
|
|---|
| Research Category |
Grant-in-Aid for General Scientific Research (C)
|
| Allocation Type | Single-year Grants |
|---|
| Research Field |
Biological pharmacy
|
|---|
| Research Institution | NATIONAL INSTITUTE OF HYGIENIC SCIENCES |
|---|
Principal Investigator |
TERAO Tadao NATIONAL INSTITUTE OF HYGIENIC SCIENCES, DIVISION HEAD, 機能生化学部, 部長 (60012605)
|
|---|
| Project Period (FY) |
1988 – 1989
|
| Project Status |
Completed (Fiscal Year 1989)
|
| Budget Amount *help |
¥2,000,000 (Direct Cost: ¥2,000,000)
Fiscal Year 1989: ¥900,000 (Direct Cost: ¥900,000)
Fiscal Year 1988: ¥1,100,000 (Direct Cost: ¥1,100,000)
|
|---|
| Keywords | Opioid / Morphine / Opioid Receptor / Mu-Receptor / Affinity Label / Phosphorylation / 〔D-Ala^2、D-Leu^2〕エンケファリンアミド / ナロキソン / ミュー受容体 |
|---|
| Research Abstract |
Tritiated morphine, DPDPE and ^<125>I-beta-endorphin were directly cross-linked to mouse brain opiate receptors by using an ultraviolet irradiation procedure. On sodium dodecyl sulfate polyacrylamide slab gel electrophoresis under reducing condition, ^3H-morphine and ^<125>I-beta-endorphin preferentially and specifically labeled a 58 kDa protein. The labeling of this protein was suppressed by the addition oil excess non-radiolabeled naloxone or beta-endorphin. ^3H-morphine and ^<125>I-beta-endorphin were covalently bound to a glycoprotein of mouse brain membranes which was retained on a wheat germ agglutinin affinity column. These results suggest that the direct UV-photoaffinity labeling method using commercially available radioactive opiates should be a useful tool for the identification of the opiate receptors.
Morphine and [D-Ala^2,D-Leu^5] enkephalinamide enhance the phosphorylation of a 58kDa protein in mouse brain synaptosomal membranes. The enhancement of phosphorylation was inhibited by naloxone, an antagonist of morphine. The phosphorylated 58 kDa protein was retained on wheat germ agglutinin-agarose and morphinone-Affi-Gel 401 columns and biospecifically eluted out from the columns with N-acetyl-D-glucosamine and naloxone, respectively. These results suggest a strong possibility that the opiate-binding protein undergoes phosphorylation by endogeous protein kinase. Since the molecular-mass of a mu-type opioid receptor in mouse brain is suggested to be 58 kDa, coincident with those of rat brain and neuroblastoma x glioma hybrid cells, it is conceivable that the phosphorylated 58 kDa protein is a mu-type receptor.
|
