鲢鱼(Hypophthal michthys molitrix)背肌中组织蛋白酶B2的纯化与酶学性质鉴定

对鲢鱼背部白肌中的组织蛋白酶B2,进行了分离纯化及酶学性质的鉴定。目的酶经粗提后,再通过酸处理、硫酸铵分级沉淀、超滤、DEAE-Sephacel、Sephacryl S-100、SP-Sepharose F.F.、Blue-sepharose 6 F.F.、Con A-Sepha-rose等步骤纯化,纯化倍数达到396.5倍。经SDS-PAGE分析,该酶呈现了分子量约为25和20KDa的2条带。组织蛋白酶B2的最适反应温度和pH分别为30~35℃和pH5.5。巯基还原剂DTT、L-Cys、β-Me均显著地激活了该酶的活性。50μmol/L的E-64能够完全抑制其活性。Cl-浓度为100μmol/L以内时,对组织蛋白酶B2起到激活作用,200μmol/L时则表现为抑制作用。25 mmol/L的P2O74-,即可有效抑制该酶的活性。该纯化酶能够水解Z-Arg-Arg-MCA、Z-Phe-Arg-MCA,但是不能够水解L-Arg-MCA。组织蛋白酶B2水解Z-Arg-Arg-MCA、Z-Phe-Arg-MCA的Km值分别为7.15及226μmol/L,表明Z-Arg-Arg-MCA是该酶的最适底物。

Purification and Characterization of Cathepsin B2 from Dorsal Muscle of Silver Carp (Hypophthalmichthys molitrix)

Silver carp cathepsin B2 was extracted from the dorsal muscle.Then it was purified to 396.5 folder by a series of procedures of acidification,ammonium sulfate precipitation,ultral-filtering,DEAE-Sephacel,Sephacryl S-100,SP-Sepharose F.F.,Blue-sepharose 6 F.F.and Con A-Sepharose.The purified enzyme assumed two bands of about 25 and 20KDa on SDS-PAGE.The optimum temperature was 30～35℃,and the optimum pH was pH 5.5.Cathepsin B2 was efficiently activated by Cys,DTT and β-ME,but was completely inhibited by E-64 of 50μmol/L.Cl-within 100μmol/L activated cathepsin B2,but when reaching 200μmol/L,it shoued effect of inhibition.P2O74-of 25mmol/L inhibited the hydrolysing activity of cathepsin B2 significantly.Substrate specificity analysis indicated that it could hydrolyze Z-Phe-Arg-MCA and Z-Arg-Arg-MCA but not L-Arg-MCA;that Z-Arg-Arg-MCA was the optimum substrate judging from the Km values of 7.15 and 226μmol/L.