1989 Fiscal Year Final Research Report Summary
OPIATE RECEPTORS: IDENTIFICATION AND FUNCTIONAL ANALYSES
| Project/Area Number |
63571071
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| Research Category |
Grant-in-Aid for General Scientific Research (C)
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| Allocation Type | Single-year Grants |
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| Research Field |
Biological pharmacy
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| Research Institution | NATIONAL INSTITUTE OF HYGIENIC SCIENCES |
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Principal Investigator |
TERAO Tadao NATIONAL INSTITUTE OF HYGIENIC SCIENCES, DIVISION HEAD, 機能生化学部, 部長 (60012605)
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| Project Period (FY) |
1988 – 1989
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| Keywords | Opioid / Morphine / Opioid Receptor / Mu-Receptor / Affinity Label / Phosphorylation |
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| 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.
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