以翡翠贻贝(Perna viridis)消化腺为材料,经GST rapTMFF柱亲和层析,分离纯化得到总谷胱甘肽硫转移酶.而后经DEAE离子交换层析得到三个洗脱峰M1、M2、M3,分别占总蛋白含量的77%,16%,2.9%,对其进行SDS-PAGE分析,结果表明M1可能是由分子量为25 kD的蛋白质亚基组成的同型二聚体,M2、M3可能为异型二聚体,M2由25kD和23 kD两个亚基组成,M3由27kD和23kD两个亚基组成.以1-氯-2,4-二硝基苯(CDNB)、3,4-二氯硝基苯(DCNB)、4-硝基氯化苄(NBC)、利尿酸(ETHA)4种底物对M1、M2、M3进行动力学分析,发现M1、M2、M3分别对ETHA、DCNB、NBC亲和力更好,Km分别为1.08mmol/L、1.51 mmol/L、0.89 mmol/L,Vmax分别为54.9μmol/min/mg,40.3 μmol/min/mg,19.4μmol/min/mg.%Total glutathione S-transferases were purified from digestive gland of mussel(Perna viridis) by GST rapTMFF.Then three glutathione S-transferase isozymes (M1, M2, M3 ) were purified and separated with ion exchange chromatography.SDS-PAGE analysis of glutathione S-transferase isozymes ( M1, M2, M3 ) indicated that M1 may be a single homodimer and the molecular weight of M1 is 25 kD, while M2 and M3 may be two heterodimers, M2 showed two bands and the molecular weight of the subunits are 25 kD and 23 kD respectively, M3 also showed two bands and the molecular weight of the subunits are 27 kD and 23 kD respectively.Enzymatic kinetic analysis of M1, M2, M3 using 1-chloro-2,4-dinitrobenzene ( CDNB ), 3,4-dichloronitrobenzene ( DCNB ), 4-nitrobenzyl chloride(NBC) and ethacrynic acid(ETHA) as substrates has a better affinity to ETHA, DCNB and NBC respectively,Km are 1.08 mmol/L, 1.51 mmol/L 1,0.89 mmol/L respectively and Vmax are 54.9 μmol/min/mg,40.3 μmol/( min · mg -1 ), 19.4 μmol/( min · mg-1 ) respectively.
展开▼
