Carr D J, DeCosta B R, Kim C H, Jacobson A E, Bost K L, Rice K C, Blalock J E
Department of Physiology and Biophysics, University of Alabama at Birmingham.
Neuroendocrinology. 1990 May;51(5):552-60. doi: 10.1159/000125390.
Opioid receptors reportedly exist on neuronal tissue of central and peripheral origin as well as on cells of the immune system. Previously, an opioid receptor has been purified from the neuroblastoma x glioma hybrid cell line, NG108-15 cells. In an effort to compare these results with opioid receptors isolated from primary neuronal tissue, we employed a methodology based on the molecular recognition theory to develop a monoclonal antibody which was used to isolate and biochemically characterize murine brain opioid receptors. We herein report the purification of an opioid receptor from mouse brain with a molecular weight of 65,000 daltons (range was 62-70 kD under reducing conditions) using a monoclonal antibody to an (the) opioid receptor. In situ labeling experiments with the delta-class selective opioid receptor affinity ligand, cis-(+)-3-methylfentanylisothiocyanate (SUPERFIT) of brain membrane confirmed these observations. Moreover, SUPERFIT, when coupled to the binding site, could block the recognition of the receptor by the monoclonal antibody. However, the selective, mu-class opioid receptor affinity reagent, 2-(p-ethoxybenzyl)-1-N,N-diethylaminoethyl-5-isothiocyanatobenz imidazole was ineffective at masking the binding site from recognition by the monoclonal antibody. Likewise, opioid-like receptors were purified from murine leukocytes which migrated at a molecular weight of 58,000 daltons under nonreducing conditions and 70,000 daltons under reducing conditions. In addition, immunoaffinity-purified receptor is shown to specifically bind the delta-class-selective opioid ligand, cis-(+)-3-methylfentanylisothiocyanate as well as the endogenous opioid peptides, beta-endorphin and [Met]-enkephalin.(ABSTRACT TRUNCATED AT 250 WORDS)
据报道，阿片受体存在于中枢和外周来源的神经组织以及免疫系统的细胞上。此前，已从神经母细胞瘤x胶质瘤杂交细胞系NG108 - 15细胞中纯化出一种阿片受体。为了将这些结果与从原代神经组织中分离出的阿片受体进行比较，我们采用了基于分子识别理论的方法来开发一种单克隆抗体，该抗体用于分离小鼠脑阿片受体并对其进行生化特性分析。我们在此报告，使用针对阿片受体的单克隆抗体，从分子量为65,000道尔顿（在还原条件下范围为62 - 70 kD）的小鼠脑中纯化出了一种阿片受体。用δ类选择性阿片受体亲和配体顺式-(+)-3-甲基芬太尼异硫氰酸盐（SUPERFIT）对脑膜进行原位标记实验证实了这些观察结果。此外，SUPERFIT与结合位点结合时，可以阻断单克隆抗体对受体的识别。然而，选择性μ类阿片受体亲和试剂2-(对乙氧基苄基)-1-N,N-二乙氨基乙基-5-异硫氰酸苯并咪唑在掩盖结合位点使其不被单克隆抗体识别方面无效。同样，从鼠白细胞中纯化出了阿片样受体，其在非还原条件下分子量为58,000道尔顿，在还原条件下为70,000道尔顿。此外，免疫亲和纯化的受体显示能特异性结合δ类选择性阿片配体顺式-(+)-3-甲基芬太尼异硫氰酸盐以及内源性阿片肽β-内啡肽和[甲硫氨酸]脑啡肽。（摘要截短于250字）
