Evans C J, Barchas J D, Esch F S, Böhlen P, Weber E
J Neurosci. 1985 Jul;5(7):1803-7. doi: 10.1523/JNEUROSCI.05-07-01803.1985.
Antibodies have been raised to a synthetic peptide corresponding to the C-terminal 15-amino acid residues of prodynorphin, the common precursor to the neo-endorphins and dynorphins. The amino acid sequence of the antigen was based on the sequence deduced from mRNA isolated and cloned from porcine hypothalamus (Kakidani, H., Y. Furutani, H. Takahashi, M. Noda, Y. Morimoto, T. Hirose, M. Asai, S. Inayama, S. Nakanishi, and S. Numa (1982) Nature 298: 245-248). Using a radioimmunoassay developed from these antibodies we have isolated an endogenous prodynorphin C-fragment from bovine caudate nucleus. The isolated peptide displayed characteristics on gel filtration similar to those of synthetic prodynorphin C-fragment predicted from the porcine mRNA sequence but had low cross-reactivity in the radioimmunoassay. Sequencing and amino acid analysis showed a substitution of serine for asparagine at position 6 in the porcine sequence. Dynorphin B (rimorphin), which is adjacent to prodynorphin C-fragment in the precursor, was isolated from the same extract. Amino acid analysis and elution position on a gel filtration column confirmed its structure as that previously characterized from bovine pituitary extracts. The release of prodynorphin C-fragment and the C-terminus of dynorphin B from the porcine precursor would require cleavage at a single arginine residue. However, a terminal arginine was not present on either of these prodynorphin peptides isolated from bovine caudate. The data would suggest that processing at a single arginine residue results in elimination of the arginine, a feature in common with processing at paired basic residues.
已制备出针对一种合成肽的抗体，该合成肽对应于强啡肽原的C末端15个氨基酸残基，强啡肽原是新内啡肽和强啡肽的共同前体。抗原的氨基酸序列基于从猪下丘脑分离并克隆的mRNA推导的序列（柿谷浩、古谷洋、高桥浩、野田美、森本洋、广濑彻、浅井正、稻山幸、中岸史、沼田修，1982年，《自然》298卷：245 - 248页）。利用由这些抗体制备的放射免疫分析法，我们从牛尾状核中分离出一种内源性强啡肽原C片段。分离得到的肽在凝胶过滤中的特性与根据猪mRNA序列预测的合成强啡肽原C片段相似，但在放射免疫分析法中的交叉反应性较低。测序和氨基酸分析表明，猪序列中第6位的天冬酰胺被丝氨酸取代。与强啡肽原C片段在前体中相邻的强啡肽B（边缘吗啡），也从同一提取物中分离出来。氨基酸分析和在凝胶过滤柱上的洗脱位置证实了其结构与先前从牛垂体提取物中鉴定的结构一致。从猪前体中释放强啡肽原C片段和强啡肽B的C末端需要在单个精氨酸残基处进行切割。然而，从牛尾状核分离得到的这两种强啡肽原肽上均不存在末端精氨酸。这些数据表明，在单个精氨酸残基处进行加工会导致精氨酸的消除，这一特征与在成对碱性残基处进行加工相同。
