PROENKEPHALIN PROCESSING

本项目的最终目标是了解 脑啡肽原和这一过程的调节。最近一段时间 肾上腺髓质脑啡肽前体已被鉴定和测序 通过DNA克隆。从这个序列和以下序列中 我们已经分离出含有脑啡肽的多肽(ECP)，这是清楚的 原脑啡肽的翻译后处理需要一个 胰酶--裂解类酶。在培养中使用嗜铬细胞我们将 使用脉冲追踪器来确定这些卵裂的顺序。我们有 在肾上腺提取液中鉴定出一种酶(S)，该酶具有最适pH 与嗜铬颗粒内的低pH值一致，并已被 显示在使用肽底物的脑啡肽原裂解部位进行裂解。 我们建议对该酶进行纯化并研究其理化性质。 它与其他蛋白酶的关系，以及它是否存在于其他 产生脑啡肽的组织，如肠道和脑。这种酶还 似乎以某种方式受到调控，因为所有可能的切割位点都是 未正常裂解，但颗粒破裂，在缓冲液中稀释 导致更大的卵裂。这一规定是如何发生的 下定决心。除了对这种酶的调节外，我们还有证据 说明含有肾上腺素的颗粒所占比例更大 脑啡肽和ECP的含量高于非肾上腺素颗粒。不管这是不是 由于胰酶样酶的调节或前脑啡肽的调节 合成将使用嗜铬细胞原代培养来确定。这个 总体结果将是理解翻译后 前脑啡肽的加工发生以及它是如何被调节以导致 正常情况下，肾上腺组织中产生ECP，神经组织中产生脑啡肽。

The ultimate goal of this project is to understand the processing of proenkephalin and the regulation of that processing. Recently the enkephalin precursor in adrenal medulla has been identified and sequenced via DNA cloning. From this sequence and the sequences of enkephalin-containing polypeptides (ECP's) we have isolated, it is clear that the post-translational processing of proenkephalin requires a trypsin-cleavage like enzyme. Using chromaffin cells in culture we will use a pulse chase to determine the order of these cleavages. We have identified an enzyme(s) in adrenal extracts, which has a pH optimum consistent with the low pH within chromaffin granules and which has been shown to cleave at proenkephalin cleavage sites using peptide substrates. We propose to purify this enzyme and study its physiochemical properties, its relationship to other proteases, and whether it is present in other enkephalin-producing tissues such as gut and brain. This enzyme also appears to be regulated in some manner as all possible cleavage sites are not cleaved normally but rupture of the granules and dilution in buffer results in much greater cleavage. How this regulation occurs will be determined. In addition to the regulation of this enzyme we have evidence suggesting that epinephrine containing granules have a greater proportion of enkephalins and ECP's than do nonepinephrine granules. Whether this is due to regulation of the trypsin-like enzyme or regulation of proenkephalin synthesis will be determined using chromaffin cell primary cultures. The overall result will be an understanding of how the post-translational processing of proenkephalin occurs and how it is regulated to result in the normally produced ECP's in adrenal tissue and enkephalins in neural tissue.