大鼠肾脏三肽基肽酶Ⅰ物理化学性质和氨基酸顺序分析  

TPP I from Rat Kideny:Its Physico-chemical Properties and Analysis of Amino Acid Sequence

作　　者：杜培革[1] 刘华欣[2] 傅桂莲[1] 大久保岩男 

机构地区：[1]北华大学医学院,中国吉林吉林132001 [2]第四军医大学吉林军医学院,中国吉林吉林132011 [3]滋贺医科大学

出　　处：《生命科学研究》2003年第2期133-138,共6页Life Science Research

基　　金：财团法人滋贺医学国际协力会资助项目

摘　　要：为进一步探讨三肽基肽酶Ⅰ(TripeptidylpeptidaseI,TPPI)的物理化性质和酶的动力学特征,通过SP 琼脂糖凝胶(SP sepharose),Phenyl cellulofine,ResorceS,超葡聚糖凝胶(SuperdexG 75)柱层析的方法,提纯大鼠肾脏三肽基肽酶Ⅰ并测定它的分子量、理化性质、酶的动力学参数及N 末端氨基酸顺序.经聚丙烯酰胺凝胶电泳分离鉴定后,得到了单一区带.提纯倍率约为8000倍.用SDS PAGE时,β 巯基乙醇(β ME)不存在和存在下,其分子量为43kD和46kD.用非变性 PAGA时,其分子量为280kD.在pH2.2～10.5范围内,TPPI具有水解Ala Ala Phe MCA的活性.该酶对于Ala Ala Phe MCA的最适pH值为3.5～4.5.在最适pH4.0条件下,对Ala Ala Phe MCA底物的Km、Vmax、Kcat和Kcat Km值分别是:680μmol L,3.7mmol (g·min),33.1 s和4.87×104 (s·mol).TPPI能被对氯汞苯甲酸盐(PCMBS)和HgCl2强烈地抑制,被二异丙基氟磷酸酯(DFP)中等程度地抑制.TPPI是受巯基试剂调节的丝氨酸酶.The physicochemical properties and kinetic parameters of tripeptidyl peptidase I(TPP I) from rat kidneys were investigated.Through successive chromatography on SPsepharose,Phenylcellulofine,Resorce S and Superdex G75 in the fast protein liquid chromatography(FPLC) column,its molecular weight,physicochemical properties and kinetic parameters as well as the Nterminal amino acid sequence were determined and analyzed.The product showed a single band by polyacrylamide gel electrophoresis (PAGE).Rat kidney TPP I was purified approximately 8 000fold.The molecular weight of the product was calculated to be approximately 43 kD and 46 kD respectively on SDSPAGE in the absence and presence of βmercaptoethanol (βME) and was 280 kD on nondenatured PAGE.The hydrolytic activity of TPP I for AlaAlaPheMCA was assayed in the pH range of 2.5～10.5.The activity toward this substrate was optimal from pH 3.5 to pH 4.5.The Km,Vmax,Kcat and Kcat/Km values of TPP I for AlaAlaPheMCA at optimal pH (4.0) were 680 μmol/L,3.7 mmol/(g·min),33.1 /s and 4.87×104/(s·mol)respectively.TPP I was strongly inhibited by PCMBS and HgCl2,and moderately by DFP.It belonging to an exotype serine peptidase regulated by SH reagent.

关 键 词：大鼠肾脏 三肽基肽酶Ⅰ 化学性质 氨基酸顺序 

分 类 号：Q556.3[生物学—生物化学]