基质辅助激光解吸／电离质谱法研究海兔卵多肽内切酶组成、结构与功能.doc

基质辅助激光解吸／电离质谱法研究海兔卵多肽内切酶组成、结构与功能
【摘要】选择DEAE52纤维素和SephadexG150为分离介质,采用柱层析法分离纯化海兔卵多肽酶（EndopeptideaseofAplysiaegg，AEE）。选用十二烷基磺酸钠聚丙烯酰胺凝胶电泳（SDSPAGE）法测定AEE纯度和亚基分子量(约39kDa)。基质辅助激光解吸／电离（MALDITOF）质谱技术测定AEE由单类型亚基(M)组成，其m/z比值分别为12738.17、18108.79和38221.42，即分子式为\[M3+\]、\[M2+\]和\[M+\]。其中AEE分子量为38221.42Da(称为AEE39)，是一种含金属锌的多肽酶。以胰岛素(INS)为探针,研究AEE39酶切INS能力,其酶切产物m/z比值为1449.51、2085.84、4080.41和4165.42。自行设计INS序列分析软件，鉴定AEE39酶切INS的位点，发现易酶切位点是LeuX(氨基酸残基)，其次是GluX，部分PhenX、AsnX和SerX结构也可以发生酶切。通过INS和海兔吸引素的分子结构比对后，指出海兔卵中的AEE39主要功能之一是负责酶切海兔吸引素，起着海兔之间信息交往、召唤、识别和交配的重要作用。AEE39具有酶切酸性多肽（acidicpeptide,AP）中LeuLeu的能力，属于一种广谱性多肽内切酶。
【关键词】海兔，多肽内切酶，质谱分析，吸引素，胰岛素
Composition,StructureandFunctionfromEndopeptideaseofAplysiaＥggＡnalyzedwithMatrixAssistedLaserDesorptionIonizationTimeofFlightMassSpectrometry
HuangHeQing,LuYongJin,LinQingMei,ZhuoHuiQin,HuangHuiYing
1(KeyLaboratoryoftheMinistryofEducationforCellBiologyandTumorCellEngineering,SchoolofLifeSciences,
2StateKeyLaboratoryofPhysicalChemistryofSolidSurface,CollageChemistry&ChemicalEngineering,
3StateKeyLaboratoryofMarineEnvironmentalScience,CollegeofOceanographyand
EnvironmentalScience,XiamenUniversity,Xiamen361005)
AbstractEndopeptideaseofAplysiaegg(AEE)wasseparatedandpurifiedbycolumnchromatographyusingseparationgelbothDEAE52cellulose.ThepurityofAEEanditsmolecularweightofthesubunit(approximately39.0kDa)weredeterminedbySephadexG150andSDSpolyacrylamidgelelectrophoresis(PAGE)methods.TheresultsfromMALDITOFmassspectrometryindicatedthatAEE39consistedofsinglesubunittype(M),anditsratioofmasstocharge(m/z)was12738.17,18108.79and38221.42,called[M3+],[M2+],and[M+],respectively.Usingprobeofinsulin(INS),acombinedtechnologywithbothmetalchelatorEDTAandMALDITOFmassspectrometrywasemployedtomeasuremolecularweightofAEE３９andtoidentifyanendopeptideasewithmetalzinc,showingmolecularweightwith38221.42Da,calledAEE39.ItwasfoundthatAEE39hasthecapacityforlysatingINS,givingenzymolysisproduceswithm/z1449.51,2085.84,4080.41,4165.42.Thesoftwaredesignedwithourselveswa