Mains R E, Eipper B A
J Biol Chem. 1979 Aug 25;254(16):7885-94.
The synthesis and secretion of various intermediate pituitary proteins was studied by using dispersed intermediate pituitary cell suspensions. Control studies indicated that the isolated cells were obtained in good yield and that after more than 24 h in culture the isolated cells continued to synthesize a collection of proteins similar to those found in freshly extracted intermediate pituitary tissue. Rat intermediate pituitary cells synthesized a molecule (Mr = 30,000; called 30K) that contained antigenic determinants for beta-endorphin, gamma-lipotropin, corticotropin (ACTH), and 16K fragment (the NH2-terminal region of mouse tumor cell pro-ACTH/endorphin). This 30K molecule, two high molecular weight forms of ACTH(13K and 20K), and 16K fragment were all shown to be glycoproteins. Continuous labeling and pulse-chase incubations were used to define the intracellular biosynthetic processing of the 30K molecule. After a 15-min pulse incubation the 30K molecule was the only labeled protein containing antigenic determinants for beta-endorphin, gamma-lipotropin, ACTH, or 16K fragment. A beta-lipotropin-like molecule served as a biosynthetic intermediate in the production of proteins similar to beta-endorphin and gamma-lipotropin. Methionine-enkephalin and alpha-endorphin were not major products in the intermediate lobe cells. Molecules similar to alpha-melanocyte-stimulating hormone and corticotropin-like intermediate lobe peptide (ACTH(18-39)) were also derived from the same 30K molecule; 20K ACTH served as a biosynthetic intermediate in this conversion. In rat intermediate pituitary cells ACTH(1-39) was not a major final product of the intracellular biosynthetic processing of the 30K molecule. The 30K molecule also served as a precursor to a protein similar to mouse tumor cell 16K fragment and related smaller proteins. With rat intermediate pituitary cells, pulse-chase experiments utilizing [35S]methionine demonstrated almost quantitative conversion of the 30K precursor into labeled proteins similar to beta-endorphin and alpha-melanocyte-stimulating hormone. In the absence of added secretagogues, small amounts of all of the smaller proteins derived from the 30K precursor were secreted coordinately into the culture medium.
利用分散的垂体中间叶细胞悬液研究了各种垂体中间叶蛋白的合成与分泌。对照研究表明，分离得到的细胞产量很高，并且在培养超过24小时后，分离的细胞继续合成一系列蛋白质，这些蛋白质与新鲜提取的垂体中间叶组织中发现的蛋白质相似。大鼠垂体中间叶细胞合成了一种分子（分子量为30,000；称为30K），该分子含有β-内啡肽、γ-促脂素、促肾上腺皮质激素（ACTH）和16K片段（小鼠肿瘤细胞前ACTH/内啡肽的NH2末端区域）的抗原决定簇。这种30K分子、两种高分子量形式的ACTH（13K和20K）以及16K片段均被证明是糖蛋白。采用连续标记和脉冲追踪孵育来确定30K分子的细胞内生物合成过程。经过15分钟的脉冲孵育后，30K分子是唯一含有β-内啡肽、γ-促脂素、ACTH或16K片段抗原决定簇的标记蛋白。一种β-促脂素样分子在产生类似于β-内啡肽和γ-促脂素的蛋白质过程中作为生物合成中间体。甲硫氨酸脑啡肽和α-内啡肽不是垂体中间叶细胞的主要产物。类似于α-黑素细胞刺激素和促肾上腺皮质激素样中间叶肽（ACTH(18 - 39)）的分子也源自同一个30K分子；20K ACTH在这种转化过程中作为生物合成中间体。在大鼠垂体中间叶细胞中，ACTH(1 - 39)不是30K分子细胞内生物合成过程的主要最终产物。30K分子还是一种类似于小鼠肿瘤细胞16K片段及相关较小蛋白质的蛋白质的前体。对于大鼠垂体中间叶细胞，利用[35S]甲硫氨酸进行的脉冲追踪实验表明，30K前体几乎定量地转化为类似于β-内啡肽和α-黑素细胞刺激素的标记蛋白。在未添加促分泌剂的情况下，源自30K前体的所有较小蛋白质都有少量被协同分泌到培养基中。
