REGULATION OF PROCESSING OF OPIOID PEPTIDE PRECURSORS

神经肽是作为大的前体蛋白合成的，它经历了 产生生物活性的特定蛋白水解酶和修饰 多肽。最初的卵裂被认为是发生在成对的或基本的 氨基酸残基与精氨酸C端赖氨酸合成肽的研究 残留物。赖氨酸或精氨酸残基被类羧肽酶去除 产生生物活性多肽的酶。尽管蛋白质水解性反应 是激活神经肽的关键步骤，我们对此知之甚少 催化这些反应的内肽酶和外肽酶。 最近，激肽释放酶丝氨酸蛋白酶家族的一个成员被 与阿片黑素皮质素原(POMC)的内切酶有关 在脑下垂体。通过使用小鼠激肽释放酶cdna探针，我们已经 能够证明单一的激肽释放酶存在于 产生POMC的小鼠垂体瘤细胞系。一种羧基肽酶 酶与牛脑啡肽原的加工有关 肾上腺髓质。该酶已纯化，并进行了部分测序。 将使用几种方法来确定激肽释放酶和 POMC和前脑啡肽需要羧肽酶 正在处理。我们将首先确定选择性移除的效果 这些蛋白质来自于细胞蛋白质池上的两个加工 先驱物。这将通过基因转移技术来完成。第二， 该酶的亚细胞和细胞分布将被确定。 第三，我们将测试这些酶的特定抑制剂的能力 改变前驱体的加工。第四，我们计划确定 编码这些酶的基因的结构。对表达的管制 这些基因的转录水平也将被分析以 确定这些基因的活性是否与 前体基因的活性。最终目标将是确定 当酶基因相对于前体基因被打开时 胚胎发育。

Neuropeptides are synthesized as large precursor proteins which undergo specific proteolytic cleavages and modifications to produce bioactive peptides. The initial cleavages are thought to occur at pairs or basic amino acid residues to produce peptides with C-terminal Lys of Arg residues. The Lys or Arg residues are removed by carboxypeptidase-like enzymes to produce bioactive peptides. Although the proteolytic reactions are critical steps in activating neuropeptides, we know very little about the endopeptidases and exopeptidase that catalyze these reactions. Recently, a member of the kallikrein family of serine proteases has been implicated in the endoproteolytic cleavage of pro-opiomelanocortin (POMC) in the pituitary. By use of a mouse kallikrein cDNA probe we have been able to demonstrate that a single kallikrein protease is present in the mouse pituitary tumor cell line that produces POMC. A carboxypeptidase enzyme has been implicated in proenkephalin processing in the bovine adrenal medulla. This enzyme has been purified and partially sequenced. Several approaches will be used to determine whether the kallikrein and carboxypeptidase enzymes are required for POMC and proenkephalin processing. We will first determine the effect of selectively removing these proteins from the cellular protein pool on processing of the two precursors. This will be done by a gene transfer technique. Second, the subcellular and cellular distribution of the enzyme will be determined. Third, we will test the ability of specific inhibitors of these enzymes to alter processing of the precursors. Fourth, we plan to determine the structure of the genes that code for these enzymes. Control of expression of these genes at the transcriptional level will also be analyzed to determine whether activity of these genes is regulated coordinately with the activity of precursor genes. A final objective will be to determine when the enzyme genes are turned on relative to the precursor genes during embryonic development.