氨氯吡咪对重组人蛋白激酶CK2全酶的抑制动力学3

CK231(1,524023)20030917,200311143,No03096;,No011766;,No2002C30109,No020101:,,48,,Tel:[phone],E2mail:[e-mail];,,39,,:Tel:[phone],Fax:[phone],E2mail:[e-mail]341;R345157;R91414;R97713;R97716A100121978(2004)0420434204CK2CK2,CK2,CK2[232P]GTP[32P]CK2CK2,IC50257157molL-1CK2:GTP,Ki236130molL-1;,Ki163163molL-1cAMPATP,GTPCK2CK2CK2;;;;IC50;CK2(),()()(22222)[1,2],CK2[1,2][3],CK2CK2[2,4],CK2HIV21,[2,4]CK2,CK2CK2,CK2[1,2]CK2,CK2,CK2,CK2[5](amiloride)[6,7],cAMPATP,GTPCK2,CK2[811],CK211.1GTPSigma,ICN,P81What2man[232P]GTP,1.21.2.1CK2CK2,[811]CK2CK2,[12]Tab11.2.2R2250,1.2.3CK2[1012]31pmol[232P]GTP32P11.2.4CK2IC50IC50[13],(probit),,IC50(50%5)434ChinesePharmacologicalBulletin2004Apr;20(4):4347©1995-2004TsinghuaTongfangOpticalDiscCo.,Ltd.Allrightsreserved.1.2.5IC50,3(080320molL-1)2gL-1,GTP612550molL-1,CK2();GTP1215molL-1,(18gL-1),CK23,Lineweaver2BurkKmVmax,CK2Ki[14]1.2.6t22.1CK2,(Tab1Fig1),CK2,,CK2,:^Y=211618X-012118,r=019861,CK2IC50257157molL-1(log214109)5,622122(DRB)N2(22)252212(A3)CK2[1,2],[12]:CK2,IC501014molL-12515molL-1,CK2,CK2Fig1EffectofamilorideonrecombinanthumanproteinkinaseCK2holoenzymeTab1EffectofamilorideonrecombinanthumanproteinkinaseCK2holoenzyme(€xs)Concentration/molL-1logvalueCK2activity/cpmInhibitoryrate%Probit0-18372478--401.6021833516450.20801.9031635074011.33.77351602.20411312124238.6334.69453202.505701445762.0335.30556402.8064167106077.4335.7388IC50=257157molL-1,33P0101vs0molL-12.2CK22.2.1GTPCK2,GTP,CK2(Fig2),3,V-1[S]-13:^Y=313413X+011120(0molL-1),^Y=413241X+011119(80molL-1),^Y=816709X+011122(320molL-1)Km2918,3816477142molL-1;Vmax8193,81948193molmin-1g-1,,Km,Vmax,,GTPCK2,Ki236130molL-1Fig2Lineweaver2BurkplotforkineticanalysisofamilorideinhibitoryeffectonrecombinanthumanCK2holoenzymeCaseinwasatfixedconcentration(2gL-1),butGTPwaschangedatindicatedconcentration.Theeachvaluerepresentsthemeanofthethreesep2arateexperiments.:control;:80molL-1;:320molL-1534ChinesePharmacologicalBulletin2004Apr;20(4)©1995-2004TsinghuaTongfangOpticalDiscCo.,Ltd.Allrightsreserved.2.2.2CK2GTP,,CK2(Fig3):3,V-1[S]-13:^Y=012056X+010560(0molL-1),^Y=013186X+010855(80molL-1),^Y=015677X+011494(320molL-1)Km3167,31733180molL-1;Vmax17186,111706169molmin-1g-1,Km,Vmax,,CK2,Ki163163molL-1Fig3Lineweaver2BurkplotforkineticanalysisofamilorideinhibitoryeffectonrecombinantCK2holoenzymeGTPwasatfixedconcentration(1215molL-1),butcaseinwaschangedatindicatedconcentration.Theeachvaluerepresentsthemeanofthethreeseparateexperiments.:control;:80molL-1;:320molL-13,Na+/H+Na+/Ca2+,[6,7]Na+/H+IC501molL-1,Na+,K+ATPaseIC503mmolL-1[6][6],cAMP(PKA,IC501mmolL-1),PKC(IC501mmolL-1),EGF(IC50350molL-1)ATP[6],(IC506molL-1),(IC502060molL-1),ATPDNA,RNA(IC50300400molL-1)[6],278283WYRFHY[15][16],[17]CK2,CK2HIV21[2,4]CK2,,,CK2[10,11],,CK2[12]CK2,ATPGTP,GTPCK2CK2,CK2,IC50257157molL-1,CK2DRBA3,EGFIC50ATP,GTPCK2,GTPCK2,CK2CK2GTP,CK2CK2,CK2CK2,30060,10,40,80()[1,2],p34cdc2,2,PKC,CK2[1,2]CK2634ChinesePharmacologicalBulletin2004Apr;20(4)©1995-2004TsinghuaTongfangOpticalDiscCo.,Ltd.Allrightsreserved.,CK2CK21LitchfieldDW.ProteinkinaseCK2:Structure,regulationandroleincel2lulardecisionsoflifeanddeath.BiochemJ,2003;369(Pt1):1152PinnaLA.ProteinkinaseCK2:Achallengetocanons.JCellSci,2002;115(Pt20):387383XuX,Landesman2BollagE,ChannavajhalaPLetal.Murineproteinki2naseCK2:geneandoncogene.MolCellBiochem,1999;191(12):65744DeClercqE.Currentleadnaturalproductsforthechemotherapyofhu2manimmunodeficiencyvirus(HIV)infection.MedResRev,2000;20(5):323495AhmedAK,AllendeJE,IssingerOG.Preface.MolCellBiochem,2001;227(12):126KleymanTR,CargoeEJJr.Amilorideanditsanalogsastoolsinthestudyofiontransport.JMembraneBiol,1988;105(1):1217KleymanTR,ShengS,KosariFetal.Mechanismofactionofamiloride:Amolecularprospective.SeminNephrol,1999;19(6):524328,,.CK2cDNA.,1999;20(2):15899,,,.CK2cDNA.,1999;15(2):2283110,,.CK2.,2000;16(1):172211,,.CK2.,2000;27(2):201512,,.AG1112CK2.,2001;17(6):6576113.,:,1985:19720214,.,:,1995:1253215IsmailovII,Kieber2EmmonsT,LinCetal.Identificationofanamiloridebindingdomainwithinthealpha2subunitoftheepithelialNa+channel.JBiolChem,1997;272(34):210758316FinchCK,KelleyKW,WilliamsRB.Treatmentoflithium2induceddia2betesinsipiduswithamiloride.Pharmacotherapy,2003;23(4):5465017SoodN,BennettWD,ZemanKetal.Increasingconcentrationofin2haledsalinewithorwithoutamiloride:effectonmucociliaryclearanceinnormalsubjects.AmJRespirCritCareMed,2003;167(2):15863InhibitorykineticsofamilorideonrecombinanthumanproteinkinaseCK2holoenzymeZHENGKe2Qin1,LIUXin2Guang,LIANGNian2Ci(InstituteofBiochemistry&MolecularBiology,1DeptofMedicalGenetics,GuangdongMedicalCollege,Zhanjiang524023)ABSTRACTAIMTostudythedirecteffectofamilorideonrecombinanthumanproteinkinaseCK2holoenzymeanditskinetics.METHODSRecombinanthumanproteinkinaseCK2andsubunitswerecloned,expressedandpurifiedbygeneengineering.Thetwosub2unitsweremixedatthesamemolarratiotoformCK2holoenzyme,whichdisplayedthemaximumbiologicalac2tivity.CK2activitywasassayedbydetectingincorporationof32Pof[232P]GTPintothesubstrateinvariouscondi2tions.RESULTSAmilorideinhi