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OPIATE RECEPTORS: IDENTIFICATION AND FUNCTIONAL ANALYSES

阿片受体：鉴定和功能分析

基本信息

批准号：
63571071
负责人：
TERAO Tadao
金额：
$ 1.28万
依托单位：
NATIONAL INSTITUTE OF HYGIENIC SCIENCES
依托单位国家：
日本
项目类别：
Grant-in-Aid for General Scientific Research (C)
财政年份：
1988
资助国家：
日本
起止时间：
1988 至 1989
项目状态：
已结题

项目摘要

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.

用紫外线照射的方法，将氚化吗啡、DPDPE和β-β-内啡肽直接与小鼠脑内阿片受体交联。在还原条件下的十二烷基硫酸钠聚丙烯酰胺平板凝胶电泳法上，~1H-吗啡和~(125)I-β-内啡肽优先和特异地标记了一个58 kDa的蛋白。这种蛋白质的标记被过量的加油抑制，非放射性标记的纳洛酮或β-内啡肽。~3H-吗啡和~(125)I-β-内啡肽与小鼠脑膜糖蛋白共价结合，该糖蛋白保留在麦芽凝集素亲和层析柱上。这些结果表明，使用市售放射性阿片类药物的直接紫外光亲和标记方法应该是鉴定阿片受体的有用工具。吗啡和[D-Ala^2，D-Leu^5]脑啡肽能促进小鼠脑突触体膜上58 kDa蛋白质的磷酸化。吗啡的拮抗剂纳洛酮可抑制这种磷酸化的增强。小麦胚胶凝集素-琼脂糖柱和吗啡酮-Affi-Gel 401柱保留了58 kDa的磷酸化蛋白，并分别用N-乙酰-D-氨基葡萄糖和纳洛酮生物特异性洗脱。这些结果表明，阿片结合蛋白很有可能通过内源性蛋白激酶发生磷酸化。由于小鼠脑内MU型阿片受体的分子质量为58 kDa，与大鼠脑和神经母细胞瘤x神经胶质瘤杂交细胞的MU型受体的分子质量一致，因此可以推测58 kDa的磷酸化蛋白是一种MU型受体。