鯉卵巢液蛋白?抑制物之研究

Abstract

本文首證實鯉魚卵巢液中含有蛋白?的抑制物，同時將之純化出來，並對其一般化學性質加以探討。 用硫酸銨沈澱法、DEAE-cellulose陰離子交換邑層分析管柱及Sephacryls-200濾篩分析法，可將鯉魚卵巢液中的蛋白?抑制物(COFPI)純化出來。其活性為237.47抑制單位／毫克，純化倍數為80，活性回收率為13%。每100毫升卵巢液可分離出3.9毫克的COFPI。 COFPI除對胰蛋白?具有強烈的抑制作用外，對於α-凝乳?亦有很強的抑制力。其分子量為50000。有SDS-聚丙烯醯凝膠電泳，可知其不具有次單元體。胺基酸組成中，以蘆荀胺酸（或蘆荀醯胺）與麩胺酸（麩醯胺）最多，共有25.8%，半胱胺酸最少，只主0.4%，氮端胺基酸?蘆荀胺酸（或蘆荀醯胺）。 COFPI對於極端酸鹼度，具有耐性，但對熱的抵抗力則不高，經攝氏175度處理30分鐘，其活性全部喪失。依動力學的研究，以酪蛋白為受質時，COFPI對胰蛋白?的抑制作用是非競爭性的，其抑制常數為2.27×10^(-7)M.

This is the first report about the presence of protease inhibitor in fish ovarian fluid. Isolation, purification and characterization of this inhibitor is reported in the present study. The purification procedures used in this study include ammonium sulfate precipitation, DEAE-cellulose column chromatography and Sephacryl S-200 gel filtration. After purification, the inhibitory activity is enhanced by 80 folds. The recovery is 13% and 3.9 mg of purified inhibitor is obtained from 100 ml of ovarian fluid. This inhibitor can inhibit both the activity of trypsin and chymotrypsin. Its molecular weight is 50000. Aspartic acid/asparagine is its N-terminal amino acid residue. The aspartic acid/asparagine and glutamic acid/glutamine are the major constituents (25.8%). This inhibitor is tolerable to extreme pH, but labile to heat treatment (loss of activity by treatment at 75℃ for 30 minutes). Its K(subscript i) is 2.27×10^(-7)M.