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Electrophoretic and irreversible heterogeneity and age-related change of enzyme molecule

酶分子的电泳和不可逆异质性以及与年龄相关的变化

基本信息

批准号：
60560084
负责人：
ICHISHIMA Eiji
金额：
$ 1.41万
依托单位：
Department of Agricultural Chemistry, Tokyo Noko University
依托单位国家：
日本
项目类别：
Grant-in-Aid for General Scientific Research (C)
财政年份：
1985
资助国家：
日本
起止时间：
1985 至 1986
项目状态：
已结题

来源：
https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-60560084/
关键词：
Serine proteinase Proteinase Molecular aging Age-related change Protease 麹菌 Aspergillus

项目摘要

A serine proteinase isolated from Aspergillus sojae seemed to be homogeneous by free boundary electrophoresis at pH 4.0 and 7.8, and the sedimentation pattern showed it to be monodisperse.However, the presence of a major band and four minor bands was noted on disc gel electrophoresis at pH 9.4. These were termed in order of their decreasing mobility towards the cathode, Sep <I> , Sep <II> , Sep <III> , Sep <IV> and Sep <V> .When angiotensin was digested with the multiple forms of serine proteinase at pH 7.5 and 30゜C, each form hydrolyzed the <Tyr^4> - <Ile^5> bond. Increasing the mobility toward the cathod from Sep <IV> to Sep <II> affects the <K_m> values only slightly, but caused a remarkable increase in the catalytic rate <k_(cat)> . When the value of <k_(cat)> / <K_m> are plotted against electrophoretic mobility of the serine proteinase toward the cathod, the curve shows age-related change like a growth curve. The maximum <k_(cat)> / <K_m> value is shown with Sep <II> .When Sep <V> was incubated in Tris-HCl buffer at pH 8.0 and 30゜C for one night, Sep <IV> was observed on SDS gel electrophoresis. Similar molecular conversion was observed with the incubation of the molecular species Sep <IV> and Sep <III> , respectively.Similar molecular weight values of these multiple forms were found on SDS gel electrophoresis. It was, however, important to learn that the SDS gel electrophoresis of Sep <I> shows a major band with molecular weight of 36 K and two lower molecular weight fraction of 19 K and 17 K when incubated at 30゜C in a pH 8.0 solution. These multiple forms have the same N- and C- terminal amino acid residues. And no significant differences and only minor conformational changes were observed.This is the first report that the catalytic activities of electrophoretic species of the serine proteinases shows age-related change.

从酱油曲霉（Aspergillussojae）中分离到一种丝氨酸蛋白酶，在pH4.0和7.8条件下的自由边界电泳显示其均一性，沉降图谱显示其为单分散性，而在pH9.4条件下的圆盘凝胶电泳则显示一条主带和四条次带。当<I><II><III><IV><V>血管紧张素被多种形式的丝氨酸蛋白酶在pH 7.5和30 ℃下消化时，每种形式都水解了<Tyr^4> - <Ile^5>键。从Sep到Sep，增加向阴极的迁移率<IV>对<II>催化速率的影响<K_m>很小，但催化速率<k_（cat）>显著增加。当<k_（cat）> /的值<K_m>与丝氨酸蛋白酶向阴极的电泳迁移率作图时，曲线显示出与年龄相关的变化，类似于生长曲线。当<K_m><II>Sep<V>在pH 8.0的Tris-HCl缓冲液中30 ℃孵育一夜后，<IV>在SDS凝胶电泳上可观察到Sep。在<IV><III>SDS凝胶电泳上发现，这两种分子形式的分子量值相似。然而，重要的是要了解，当在<I>pH 8.0的溶液中于30 ℃下孵育时，Sep的SDS凝胶电泳显示分子量为36 K的主带和两个较低分子量部分19 K和17 K。这些多种形式具有相同的N-和C-末端氨基酸残基。这是首次报道丝氨酸蛋白酶电泳活性随年龄变化的现象。