PROENKEPHALIN IN PROCESSING IN THE ADRENAL MEDULLA

本项目的最终目标是了解 脑啡肽原和对这种加工的调节。 最近 肾上腺髓质中的脑啡肽前体已被鉴定并测序 通过DNA克隆。 从这个序列和 脑啡肽多肽（ECP的），我们已经分离，很明显， 脑啡肽原的翻译后加工需要一个 胰蛋白酶裂解样酶。 利用培养的嗜铬细胞， 使用脉冲追踪来确定这些分裂的顺序。 我们有 在肾上腺提取物中鉴定了一种酶，其具有最适pH值 与嗜铬颗粒内的低pH值一致， 显示使用肽底物在脑啡肽原切割位点切割。 我们建议纯化该酶并研究其理化性质， 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，如肠和脑。 这种酶也 似乎以某种方式受到调节，因为所有可能的切割位点都是 未正常裂解，但颗粒破裂并在缓冲液中稀释 导致更大的分裂。 这一规则将如何发生， 测定 除了对这种酶的调节，我们有证据表明 这表明含有肾上腺素的颗粒具有更大的比例 脑啡肽和ECP比非肾上腺素颗粒更强 这是否是 由于胰蛋白酶样酶的调节或脑啡肽原的调节 将使用嗜铬细胞原代培养物测定合成。 的 总体结果将是理解翻译后如何 脑啡肽原的加工发生，以及它是如何被调节，从而导致 正常情况下，肾上腺组织产生ECP，神经组织产生脑啡肽。

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.