一种分析叶绿体类囊体膜色素蛋白复合物的蓝绿温和胶电泳系统_李贝贝

＊李贝贝1)　郭进魁1)　周　云1)　张珠珠1)　张立新1,2)＊＊(1),730000;2),730000)　,PSⅠ,PSⅡ,ATP,b6f,1,5-.SDS-50,,、.　,,,　Q946　　,,CO2.,1,5-(RuBPase)Ⅰ(PSⅠ),Ⅱ(PSⅡ),ATP,b6f.,,[1].、,.SDS/,(CP),[2,3].1991,Sch‐gger[4],,blue-nativepolyacrylamidegelelectrophoresis(BN-PAGE).,、、.,,,,[4～6].,,,.,.1　1.1　1g/LHgCl230min,2h,24h.7,120μmol·m-2·s-1.1.2　710g,150ml330mmol/L,2mmol/LEDTA-KOH,1mmol/LMgCl2,50mmol/LHepes-KOH,pH7.7,5mmol/L,2,1000g2min,330mmol/L,50mmol/LHepes-KOH,pH8.0,1mmol/LDTT(SH),35%,70%Percoll,4500g5min,.1300g2min,SH,1000g2min,[7].50mmol/LHepes-KOH,pH8.0,1mmol/LMgCl2,14000g5min,.1.3　Chl0.4g/L,0.5%,30min,　＊,(199924).　＊＊.　Tel:[phone],E-mail:[e-mail]　:2002-12-24,:2003-02-27·639·2003;30(4)　　　　　　　　　Prog.Biochem.Biophys.5mmol/LMgCl2,50mmol/LHepes-KOH,pH8.0,10mmol/LNaF10,4000g5min,10000g10min,.40000g30min,100000g90min,[6].1.4　1.4.1　:,,,5%～13.5%,4%[3].1.Table1　Solutionforthepreparationof5%～13.5%gradientbluenativegelStacking(4%)Separation(5%)Separation(13.5%)480g/Lacrylamideand15g/Lbisacrylamide0.807ml0.707ml1.91ml150mmol/LBisTris,1.5mol/L6-amino-caproicacid,pH7.03.33ml2.33ml2.33ml750g/Lglycerol00.47ml1.870mlH2O5.733ml3.427ml0.823mlTEMED20μl7.0μl7.0μl50g/LAPS100μl40.0μl40.0μlTotal10ml10ml10ml1.4.2　:25mmol/LBisTris-HCl,20%,14000g5min,,14000g10min,1%ServaG,.、1%ServaG.1.4.3　:50mmol/LTricine,15mmol/LBisTris,pH7.050mmol/LBisTris-HCl,pH7.0.4℃.1.5　SDS--,(8mol/L,5%SDS,20%,10%,50mmol/LTris-HCl,pH6.8)30min,,SDS--.,25mA.1.6　SDS--,Sambrook[8],5%,10mmol/LTris-HCl,pH7.5,150mmol/LNaCl0.05%Tween201h,.1h,NBTBCIP.D1,LHCⅡ,PsaA/B,RubisCODr.Satoh,Dr.Staehelin,Dr.GrayDr.Muto.2　　　2.1　1,Fig.1　Blue-nativegelelectrophoresisanalysisofpeathylakoidmembraneproteincomplexes(a)Thylakoidmembranesweresolubilizedby0.5%(1),1%(2)and2%(3)DMinthepresenceofChl0.5g/L,and0.5%(4),1%(5)and2%(6)DMinthepresenceofChl1g/L.(b)Thylakoidmembranesweresolubilizedby0.5%(1),1%(2)and2%(3)digitonininthepresenceofChl0.5g/L,and0.5%(4),1%(5)and2%(6)digitonininthepresenceofChl0.5g/L.·640·　　Prog.Biochem.Biophys.　　　　　　　　2003;30(4).,,,[5].(DM),.BN-PAGE,100ku600ku.Chl0.5g/L1%DM,RuBPb6f,6(1a).,,,(1b).2.2　,SDS--.,,.1%DM,Chl0.5g/LBN,,(2b).BN,SDS--,RuBP、LHCⅡ、PSⅠ、D1.PSⅠ540ku,,PSⅠPsaA/B.[1],80kuPsaAPsaB,21～25kuLHCⅠ20ku.450kuPSⅠ,540kuPSⅠ,PSⅠLHCⅠ.RuBP430ku,,,2,53ku13ku.LHCⅡ,180ku.D1260ku230kuPSⅡ,SDS--:260kuPSⅡ,CP47、CP43、D2D1;230kuCP43PSⅡ.Fig.2　TwodimensionalanalysisofproteincompositionofthylakoidmembranepigmentproteincomplexseparatedbybluenativegelelectrophoresisandimmunoblottingidentificationAlaneofablue-nativegel(a)wascutoutandtransferredhorizontallyonaSDS-urea-polyacrylamidegelforelectrophoresisintheseconddimension(b).Thetwo-dimensionalseparatedproteinswereelectroblottedontoNCmembranesandimmunodetectedwithantibodiesofagainstspecificproteincomponentsofthylakoidmembraneproteincomplexes(c).2.3　;PSⅠ,PSⅡ[9].,Chl0.5g/L,1%DM,.,,PSⅠPSⅡ[9].,SDS--.(3).·641·2003;30(4)　　　　　　　　　Prog.Biochem.Biophys.Fig.3　AnalysisofstromalthylakoidandgranalthylakoidmembraneproteincomplexesThestromalthylakoidandgranalthylakoidmembraneproteincomplexes(a:1,2)weresolubilizedby1%DM,thenlanesofblue-nativegelwereplacedonurea-SDSgelsandtwo-dimensionalmapofthylakoidmembraneproteincomplexesareshown(b,c).3　　　,.,,,,[3,10].,,,,,,(1).Ⅰ、Ⅱ、b6f、Ⅱ、ATPRuBP.,(2).(3),[9].,、..,,ⅡPSⅡ,PSⅡ.PSⅡ,CP43[4～6].PSⅠ,PSⅠ[11].,,、,.,,、.　　　1　WollmanFA,MinaiL,NechushtaiR.Thebiogenesisandassemblyofphotosyntheticproteinsinthylakoidmembranes.BiochimBiophysActa,1999,1411(1):21～812　,,,..,1995,22(3):268～272DuLF,LinHH,PanYH,etal.ProgBiochemBiophys,1995,22(3):268～2723　PeterGF,ThornberJP.ElectrophoreticproceduresforfractionationofPhotosystemsⅠandⅡpigment-proteinsofhigherplantsandfordeterminationoftheirsubunitcomposition.MethPlantBiochem,1991,5:195～2104　Sch‐ggerH,vonJagowG.Bluenativeelectrophoresisforisolationofmembraneproteincomplexesinenzymaticallyactiveform.AnalBiochem,1991,199(2):223～2315　KǜglerM,J‐nschL,KruftV,etal.Analysisofthechloroplastproteincomplexesbyblue-nativepolyacrylamidegelelectrophoresis(BN-PAGE).PhotosynRes,1997,53(1):35～446　ZhangL,PaakkarinenV,SuorsaM,etal.ASecYhomologueisinvolvedinchloroplast-encodedD1proteinbiogenesis.JBiolChem,2001,276(41):37809～378147　ZhangL,PaakkarinenV,vanWijkKJ,etal.Co-translationalassemblyoftheD1proteinintoPhotosystemⅡ.JBiolChem,1999,274(23):16062～160678　SambrookJ,RussellDW.MolecularCloning:ALaboratoryManual.3rd.NewYork:ColdSpringHarborLaboratoryPress,2001.18.48～18.549　WollenbergerL,StefanssonH,YuSG,etal.Isolationandcharacterizationofvesiclesoriginatingfromthechloroplastgranamargins.BiochimBiophysActa,1994,1184(1):93～102·642·　　Prog.Biochem.Biophys.　　　　　　　　2003;30(4)10AndersonB,AnderssonJM,RyrieIJ.Transbilayerorganizationofthechlorophyll-proteinsofspinachthylakoids.EurJBiochem,1982,123(2):465～47211,.Ⅰ.,1998,43(18):1973～1977ZhengCX,GaoFR.ChinSciBull,1998,43(18):1973～1977BlueNativeGelElectrophoresisAnalysisofChloroplastPigmentProteinComplexes＊LIBei-Bei1),GUOJin-Kui1),ZHOUYun1),ZHANGZhu-Zhu1),ZHANGLi-Xin1,2)＊＊(1)SchoolofLifeScience,LanzhouUniversity,Lanzhou730000,China;2)ColdandAridRegionsEnvironmentalandEngineeringResearchInstitute,TheChine