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1、MethodsinMolecularBiologyMethodsinMolecularBiologyTMVOLUME116ProteinProteinLipidationLipidationProtocolsProtocolsEditedbyEditedbyMichaelH.GelbMichaelH.GelbHUMANAPRESSHUMANAPRESSInVitroAnalysisofGPIBiosynthesis11InVitroAnalysisofGPIBiosynthesisinMamma
2、lianCellsVictoriaL.Stevens1.Introduction1.1.BackgroundThebasicstrategyusedinmostassaysofactivitiesinvolvedinthebiosyn-thesisofglycosylphosphatidylinositol(GPI)inmammaliancellsisthesameasisemployedforotherlipidbiosyntheticpathways.Thatis,radioactivity
3、istrans-ferredfromawater-solublesubstrateintoalipophilicproduct.Afterthereac-tioniscomplete,thedifferentialsolubilityofthesubstrateandproduct(s)isexploitedtoseparatetheseradiolabeledcompounds.InGPIbiosynthesis,atleastoneofthesubstratesineachstepandal
4、loftheenzymesinthepathwayaremembrane-associatedandlocalizedtotheendoplasmicreticulum.There-fore,multipleGPIbiosyntheticactivities,aswellassomeofthesubstratesforlaterstepsinthepathway,arepresentinthecellularpreparationsusedintheassays.Forthisreason,mu
5、ltipleintermediatesinGPIbiosynthesisareusuallygeneratedinasinglereaction.Althoughitispossibletooptimizetheassayconditionsforonestep,itisusuallyimpossibletostudyonereactionindepen-dentlywiththistypeofcell-freesystem.AssaysforindividualreactionsinGPIbi
6、osynthesisarepossibleifsyntheticGPIintermediatesareavailable.Todate,thesecondandthirdreactionsinthepathwayhavebeenmeasuredinmammaliancellswithexogenouslysuppliedGlcNAc-PI(1)andGlcN-PI(2),respectively.Inthelattercase,ashort-chain(dioctanoyl)analogofth
7、eGPIintermediatewasused.Becausetheshort-chainanalogsaremorewater-soluble,theyaremucheasiertodelivertothemem-branesusedasasourceoftheGPIbiosyntheticactivities.From:MethodsinMolecularBiology,Vol.116:ProteinLipidationProtocolsEditedby:M.H.Gelb©HumanaPre
8、ssInc.,Totowa,NJ12StevensFig.1.GPIbiosynthesisinmammalsandyeast.1.1.1.PathwayforGPIBiosynthesisinMammalianCellsThebiosynthesisofGPIproceedsbythesequentialadditionofcarbohy-dratestophosphatidylinositol(PI),asisshowninFig.1(reviewedinrefs.3and4).Inthef
