摘要
棕尾别麻蝇幼虫肠液经ＳＤＳ－ＰＡＧＥ后，Ｘ光片显影，呈现两条蛋白酶活性带．ＩＥＦ后，两条蛋白酶活性带的等电点分别为ｐＨ７．７和６．８．麻蝇幼虫肠液经５５％～７５％硫酸铵沉淀，以及连续两次制备等电聚焦，分离纯化出等电点约为ｐＨ７．７，分子量约为３５ｋＤ的蛋白酶ＢＧＰ．该酶能分解酪蛋白和类胰蛋白酶专一底物Ｂｚ－Ｐｈｅ－Ｖａｌ－ＡｒｇＮＡ，不能分解弹性蛋白酶专一底物ｅｌａｓｔｉｎ－ＣｏｎｇｏＲｅｄ和类胰凝乳蛋白酶专一底物Ｓｕｃ（Ａｌａ）２Ｐｒｏ－ＰｈｅＮＡ．ＳＢＢＩ，Ｌｅｕｐｅｐｔｉｎ和ＰＭＳＦ能强烈抑制其活性．专一底物和抑制剂的结果表明，ＢＧＰ是一种类胰蛋白酶．其最适反应温度为５０℃，最适作用ｐＨ为８．５．不耐高温，５０℃保温３０ｍｉｎ活性急剧下降．Ｈｇ２＋，Ｚｎ２＋和Ｃｕ２＋能抑制酶活性．Ｃａ２＋，Ｍｇ２＋对酶无激活作用，ＥＤＴＡ无抑制作用．
The proteolytic activity present in the gut of larvae of Boettcherisca peregrina was detected.Separation of the whole intact gut extract by SDSPAGE and subsequent incubation with Xray film visualized two separate protease activity bands.After separated by IEF,two separate protease activity bands were also shown with isoelectric point 7.7 and 6.8 respectively.A protease BGP (Boettcherisca pergrina gut protease,pI 7.7,Mr 35 kD)was purified to homogeneity after 55%—75% ammonium sulfate precipitation and two times preparative isoelectric focusing continuously by Rotofor Cell IEF system.BGP showed strong activity against casein and BzPheValArg NA(specific substrate for trypsin).It possessed no activity against elastinCongo Red and Suc(Ala)2ProPhe NA(specific substrate for elastase and chymotrypsin,respectively).SBBI,Leupeptin and PMSF showed strong inhibition on BGP.Substrate specificity and inhibition by specific inhibitors demonstrated that BGP was a trypsinlike enzyme.Metal ions Hg2+,Zn2+and Cu2+ could inactivate BGP strongly.However,Mg2+and Ca2+ could not increase its activity.BGP was most active at 50℃ and pH 8.5.It was sensitive to temperature and its activity decreased drastically after incubation at 50℃ for 30 min.
出处
《中国生物化学与分子生物学报》
CAS
CSCD
1998年第4期402-406,共5页
Chinese Journal of Biochemistry and Molecular Biology
关键词
麻蝇
蛋白酶
分离纯化
性质
Boettcherisca peregrina,Protease,Purification and characterization
