牛嗜铬颗粒中内蛋白水解活性对重组脑啡肽前体的切割作用。

Cleavage of recombinant enkephalin precursor by endoproteolytic activity in bovine chromaffin granules.

作者信息

Hook V Y, Hegerle D, Affolter H U

机构信息

Department of Biochemistry, Uniformed Services University of the Health Sciences, Bethesda, MD 20814.

出版信息

Biochem Biophys Res Commun. 1990 Mar 16;167(2):722-30. doi: 10.1016/0006-291x(90)92085-e.

DOI:10.1016/0006-291x(90)92085-e

PMID:2182020

Abstract

To identify endoproteolytic activity that processes the enkephalin precursor, a novel approach was undertaken for the production of model substrate in the form of recombinant 35S-(Met)-preproenkephalin (35S-(Met)-PPE), generated by in vitro transcription and translation of the rat PPE cDNA. Endoproteolytic activity in bovine chromaffin granules cleaved 35S-(Met)-PPE with a pH optimum of 4.5 and generated multiple products containing the NH2-terminal segment of the precursor. Processing of 35S-(Met)-PPE, as well as endogenous enkephalin intermediates, was inhibited by pepstatin A and stimulated by DTT. These results suggest that aspartyl and thiol proteolytic activity(ies) are involved in cleaving the enkephalin precursor.

摘要

为鉴定加工脑啡肽前体的内蛋白水解活性，采用了一种新方法来生产重组35S-（甲硫氨酸）-前脑啡肽原（35S-（甲硫氨酸）-PPE）形式的模型底物，该底物通过大鼠PPE cDNA的体外转录和翻译产生。牛嗜铬颗粒中的内蛋白水解活性以pH 4.5为最适条件切割35S-（甲硫氨酸）-PPE，并产生多种含有前体NH2末端片段的产物。胃蛋白酶抑制剂A抑制35S-（甲硫氨酸）-PPE以及内源性脑啡肽中间体的加工，而二硫苏糖醇则刺激其加工。这些结果表明，天冬氨酰和巯基蛋白水解活性参与了脑啡肽前体的切割。