Madden J, Evans C J, Tyler A N, Esch F S, Böhlen P, Makk G, Weber E
Department of Psychiatry and Behavioral Sciences, Stanford University School of Medicine, California 94305.
J Neurochem. 1991 Jun;56(6):1914-20. doi: 10.1111/j.1471-4159.1991.tb03448.x.
The rabbit cerebellum has been shown to contain significant quantities of opioid receptors consisting of both mu- and kappa-subtypes. To determine the nature of the endogenous opioid ligands in this tissue, extracts from rabbit cerebellum were separated by various chromatography techniques and fractions were assayed initially for opioid peptides with a radioimmunoassay capable of detecting all peptides with an amino-terminal Tyr-Gly-Gly-Phe sequence. This sequence is common to all mammalian opioid peptides and is critical for recognition by all known opioid receptors. Each of the three immunoreactive opioid peptide peaks detected was purified to homogeneity and subjected to amino acid composition and sequence analysis. One peak was analyzed further by mass spectrometry. This identified the major opioid peptides in the cerebellum as [Met5]enkephalin, [Leu5]enkephalin, and heptapeptide [Met5]enkephalyl-Arg6-Phe7. The comprehensiveness of this initial detection scheme in identifying biologically active opioid peptides was substantiated through subsequent analysis. Using specific radioimmunoassays for representative opioid peptides of the three opioid systems currently known, no other peptides of either the proenkephalin, proopiomelanocortin, or prodynorphin series were detected in any appreciable amounts. Collectively, these results are consistent with the position that rabbit cerebellar opioids are derived from proenkephalin. However, given that no appreciable quantities of either [Met5]enkephalyl-Arg6-Arg7-Val8-NH2 (metorphamide) or [Met5]enkephalyl-Arg6-Gly7-Leu8 were detected suggests that rabbit proenkephalin may have a slightly altered sequence and/or is differentially processed relative to other mammalian species studied.
已证明兔小脑含有大量由μ和κ亚型组成的阿片受体。为了确定该组织中内源性阿片配体的性质，用各种色谱技术分离兔小脑提取物，并用一种能够检测所有具有氨基末端Tyr-Gly-Gly-Phe序列的肽的放射免疫分析法对各组分进行阿片肽初步检测。该序列是所有哺乳动物阿片肽共有的，对所有已知阿片受体的识别至关重要。检测到的三个免疫反应性阿片肽峰中的每一个都被纯化至同质，并进行氨基酸组成和序列分析。其中一个峰通过质谱进一步分析。这确定小脑中的主要阿片肽为[Met5]脑啡肽、[Leu5]脑啡肽和七肽[Met5]脑啡肽-Arg6-Phe7。通过后续分析证实了这种初始检测方案在鉴定生物活性阿片肽方面的全面性。使用针对目前已知的三种阿片系统的代表性阿片肽的特异性放射免疫分析法，未检测到前脑啡肽、促肾上腺皮质激素原或前强啡肽系列的任何其他肽有可观的量。总体而言，这些结果与兔小脑阿片肽源自前脑啡肽的观点一致。然而，鉴于未检测到可观量的[Met5]脑啡肽-Arg6-Arg7-Val8-NH2（甲硫啡肽酰胺）或[Met5]脑啡肽-Arg6-Gly7-Leu8，这表明兔前脑啡肽的序列可能略有改变和/或相对于其他研究的哺乳动物物种有不同的加工方式。
