MMB 206 Peptide Research

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時(shí)間:2019-07-20

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1、MethodsinMolecularBiologyMethodsinMolecularBiologyTMVOLUME206PeptideResearchPeptideResearchProtocolsProtocolsEndothelinEndothelinEditedbyEditedbyJanetJ.MaguireJanetJ.MaguireAnthonyP.DavenportAnthonyP.DavenportHUMANAPRESSHUMANAPRESSRadioligandBindingAssays454Radiolig

2、andBindingAssaysandQuantitativeAutoradiographyofEndothelinReceptorsAnthonyP.DavenportandRhodaE.Kuc1.Introduction1.1.EndothelinReceptorsEndothelin(ET)receptorsbelongtotheG-protein-linkedseventransmem-branespanningfamilyofreceptors,locatedintheplasmamembrane.Recep-tor

3、scanbeidentifiedbytheiraminoacidstructureandprovideunambiguousevidenceforexpressionofageneencodingaparticularsub-typeinspecificcellsortissues(seeChapter6).Todate,onlytwoETreceptorsub-typeshavebeenisolatedandclonedfrommammaliantissues,ETA(1)andETB(2).ETreceptorsareun

4、usualinbeingisolatedandclonedbeforethediscoveryofsub-typeselectiveantagonists.However,receptorfunctioncannotbepredictedfromtheaminoacidsequencealone.Thetwosub-typeswereoriginallydistinguished,usingfunctionalstud-ies,bytheirrankorderofaffinityfortheendogenouspeptides

5、:ET-3typicallydisplaysatleasttwoordersofmagnitudeloweraffinityfortheETAreceptorthanET-1,whereasbothpeptidesareequipotentattheETBreceptor(3,seeTable1).CharacterizingthefunctionalroleofETreceptorsubtypesbyinvitroassaysandestablishingwhethersyntheticcompoundsareselecti

6、veagonistsorantagonistsisdescribedinChapters5and11.Whilefunctionalstudiescanprovidequantitativemeasurementofaffinities,ligand-bindingassaysareneededtomeasureasecondkeyparameter,receptordensity,andcombinedwithquantitativeautoradiography,canbeusedtovisualizethedistrib

7、utionofreceptorswithintissue.Thecombinationofmoleculartechniqueswithligandbindinginthesametissuecanprovidecompellingevidenceforreceptorexpression(4,5).From:MethodsinMolecularBiology,vol.206:PeptideResearchProtocols:EndothelinEditedby:J.MaguireandA.Davenport?HumanaPr

8、essInc.,Totowa,NJ4546DavenportandKucTable1ClonedMammalianEndothelinReceptorsETAETBETB2ETC(mammalian)(mammalian)(avian)(amphibian)PotencyET

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