BIOSYNTHESIS, PROCESSING & SECRETION OF NEUROPEPTIDES & PITUITARY PEPTIDE HORMONE

脑垂体腺泡酶参与果胶原的加工 对阿片黑素皮质素(POMC)、促肾上腺皮质激素原/内啡肽进行了研究。一个7万摩尔。 WT.Ca++激活配对碱性残基特异的天冬氨酸蛋白酶 的POMC(POMC转换酶PCE)已经得到了足够的纯化 用于微测序的数量。一种酵母天冬氨酸蛋白酶，由 处理亲α交配因子的YAP3基因过表达 在缺乏天冬氨酸蛋白酶的酵母菌株中。YAP3酶已经被 纯化并证明具有特异性、抑制物轮廓、大小和 免疫学特性类似于哺乳动物的PCE。PCE似乎是 YAP3酶的哺乳动物同源物。YAP已经与 GH3细胞中的POMC。YAP3被证明在这些细胞中处理POMC在 与纯化后的酶在体外的反应方式相同。一种酸性的钙离子 ACTH四碱性位点特异性激活的丝氨酸蛋白酶 已被分离并部分提纯。一种PC2样丝氨酸蛋白酶 在成对的碱性残基和四碱基残基上切割POMC ACTH已在牛的分泌囊泡膜和 部分提纯。一种能裂解的单/二肽基羧肽酶 ACTH1-39至ACTH1-38和ACTH1-37已被鉴定、表征和 牛中间叶分泌囊的部分纯化 膜。POMC靶向受调控细胞的信号 并对其分泌途径进行了研究。截断的POMC/CAT构造 将其导入AtT-20细胞后，发现其前26个氨基酸残基 形成由两个二硫键稳定的发夹环的POMC 包含将前荷尔蒙靶向受调控的 分泌途径。带有N-末端发夹环的POMC突变体 在被移除的二硫键中，没有被靶向分泌 颗粒。这些结果表明独一无二的 大豆POMC N-末端的构象和二硫键 促进这种前激素靶向分泌颗粒。

Pituitary secretory vesicle enzymes involved in the processing of pro- opiomelanocortin (POMC, pro ACTH/endorphin) were studied. A 70,000 mol. wt. Ca++ activated aspartic protease specific for paired basic residues of POMC (POMC converting enzyme PCE) has been purified in sufficient quantities for microsequencing. A yeast aspartic protease encoded by the YAP3 gene which processes pro-alpha-mating factor has been overexpressed in a yeast strain lacking aspartic proteases. The YAP3 enzyme has been purified and shown to have specificity, inhibitor profile, size and immunological properties similar to the mammalian PCE. PCE appears to be the mammalian homologue of YAP3 enzyme. YAP has been co-expressed with POMC in GH3 cells. YAP3 was shown to process POMC in these cells in a manner identical to the purified enzyme in vitro. An acidic, Ca++ activated serine protease specific for the tetrabasic sites of ACTH has been isolated and partially purified. A PC2-like serine protease which cleaves POMC at paired basic residues and the tetrabasic residue site of ACTH has been identified in bovine secretory vesicle membranes and partially purified. A mono-/dipeptidyl carboxypeptidase which cleaves ACTH1-39 to ACTH1-38 and ACTH1-37 has been identified, characterized and partially purified from bovine intermediate lobe secretory vesicle membranes. The signal involved in the targeting of POMC to the regulated secretory pathway was also investigated. Truncated POMC/CAT constructs transfected into AtT-20 cells indicated tat the first 26 amino acids of POMC which forms a hairpin loop stabilized by two disulfide bridges contains the signal for targeting the prohormone to the regulated secretory pathway. POMC mutants with the N-terminal hairpin loop, or one of the disulfide bridges removed failed to be targeted to secretory granules. These results indicate the importance of the unique conformation and disulfide bridges of the N-terminus of POMC in facilitating targeting of this prohormone to secretory granules.