Eipper B A, Park L, Keutmann H T, Mains R E
J Biol Chem. 1986 Jul 5;261(19):8686-94.
Based on sequence data, rat and mouse pro-adrenocorticotropin (ACTH)/endorphin could give rise to joining peptide, a short acidic peptide that could terminate with a glutamic acid alpha-amide. Rat and mouse pituitary cells were found to cleave the pro-ACTH/endorphin precursor at an -Arg-Arg- site to produce primarily joining peptide-sized material. The amounts of joining peptide were approximately equimolar to the other major pro-ACTH/endorphin-derived products. Using antisera specific for the COOH-terminal modifications of joining peptide and three analytical approaches which separate amidated from glycine-extended forms of joining peptide, it was found that most of the joining peptide in murine anterior and intermediate pituitary was amidated. Identification of the amidated and glycine-extended forms of joining peptide was confirmed by amino acid analysis of the purified molecules. When anterior pituitary corticotrope tumor cells were grown in culture medium lacking ascorbate, there was no detectable ascorbate in the cells; nevertheless, a significant fraction of the joining peptide produced was alpha-amidated, indicating that production of alpha-amidated product was not totally dependent on ascorbate. The amidation state of the joining peptide produced by mouse corticotrope tumor cells was responsive to added ascorbate. Cells grown in medium containing ascorbic acid at the levels found in plasma concentrated the ascorbate to the levels normally found in pituitary tissue, and nearly all of the joining peptide produced was alpha-amidated. The amidation state of secreted joining peptide mirrored the amidation state of the joining peptide in the cells.
根据序列数据，大鼠和小鼠的促肾上腺皮质激素（ACTH）/内啡肽前体可产生连接肽，一种可由谷氨酸α-酰胺终止的短酸性肽。研究发现，大鼠和小鼠的垂体细胞在-Arg-Arg-位点切割促肾上腺皮质激素/内啡肽前体，主要产生连接肽大小的物质。连接肽的量与其他主要的促肾上腺皮质激素/内啡肽衍生产物大致等摩尔。使用针对连接肽COOH末端修饰的抗血清以及三种将连接肽的酰胺化形式与甘氨酸延伸形式分离的分析方法，发现小鼠垂体前叶和中叶中的大多数连接肽都被酰胺化。通过对纯化分子的氨基酸分析证实了连接肽的酰胺化形式和甘氨酸延伸形式的鉴定。当垂体前叶促肾上腺皮质激素肿瘤细胞在缺乏抗坏血酸的培养基中培养时，细胞中未检测到抗坏血酸；然而，产生的连接肽中有很大一部分被α-酰胺化，这表明α-酰胺化产物的产生并非完全依赖于抗坏血酸。小鼠促肾上腺皮质激素肿瘤细胞产生的连接肽的酰胺化状态对添加的抗坏血酸有反应。在含有血浆中发现水平的抗坏血酸的培养基中生长的细胞将抗坏血酸浓缩到垂体组织中通常发现的水平，并且产生的几乎所有连接肽都被α-酰胺化。分泌的连接肽的酰胺化状态反映了细胞中连接肽的酰胺化状态。
