Fourmethods(anenzymeactivityassay,westernblotting,RT-PCR,andnorthernhybridization)todetecttheenzymeD-amino-acidoxidasearedescribed.

D-Amino-acidoxidasecatalyzesoxidativedeaminationofD-aminoacids(stereoisomersofnaturallyoccurringL-aminoacids)tothecorresponding2-oxoacids,producingammoniaandhydrogenperoxideinthecourseofthereaction(1).Thisenzymeispresentinawidevarietyoforganisms(2).However,thephysiologicalroleofthisenzymehasbeenunclearbecauseitssubstrateD-aminoacidshavebeenconsideredtoberareineukaryoticorganisms(2,3).Inpreviouswork,mycolleaguesandIconcludedthatD-amino-acidoxidaseisnotpresentinthemouseliver(13).Thispaperexaminesingreaterdetailthemethodsusedinreachingthatconclusion.

AssayofD-amino-acidoxidaseactivity

D-Amino-acidoxidaseactivitycanbemeasuredbyvariousmethods.OneofthemisthecolorimetricmethodofWatanabeetal.(4).Organs,tissues,ortissueculturecellswerehomogenizedin7mMpyrophosphatebuffer(pH8.3).Thehomogenateswerecentrifugedat550xgfor5minutes.Thesupernatantsolutionswereusedfortheassay.Thereactionmixtureconsistedof0.3mlof0.133Mpyrophosphatebuffer(pH8.3)containingcatalaseat700IU/ml,0.3mlof0.1MD-alanine,0.2mlof0.1mMFAD,and0.1mlof70%(V/V)methanol.Thereactionwasstartedbytheadditionof0.1mlofthesupernatantsolution.Thereactionwascarriedoutat37°Cfor15-60minutesdependingontheactivityofthesamples.Itwasterminatedbytheadditionof1mlof10%trichloroaceticacid.Inablank,trichloroaceticacidwasaddedtothereactionmixturebeforetheenzymereactionwasstarted.Theprecipitatewasremovedbycentrifugation(700xg,20min).To0.5mlofthesupernatantsolutionwereadded0.5mlof5NKOHand0.5mlof0.5%4-amino-3-hydrazino-5-mercapto-1,2,4-triazolein0.5NHCl.Themixturewaskeptstandingatroomtemperaturefor15min.After0.5mlof0.75%KIO₄in0.2NKOHwereaddedtothemixturewithvigorousshaking,absorbanceat550nmwasD-Amino-acidoxidaseactivityiscalculatedwiththisformula:Activity(μmolmin^-1=2.584A/t,whereAisadifferentialabsorbanceat550nmbetweenthesampleandtheblank,andtisthereactiontimeinminutes.Thisactivityvaluewasfurtherdividedbythequantityoftheproteinpresentinthefirstreaction.TheproteinconcentrationinthesupernatantsolutionwasdeterminedaccordingtothemethodofLowryetal.(5)usingbovineserumalbuminasastandard.ItwasalsodeterminedusingaProteinAssayKit(Bio-Rad,Hercules,CA).D-Amino-acidoxidaseactivityisfinallyexpressedastheamountofD-alanineoxidizedperminpermilligramofprotein.HogkidneyD-amino-acidoxidase(Sigma,St.Louis,MOorBoehringerMannheim,Germany)wasusedasacontrol.

WesternBlotting

Organs,tissues,ortissueculturecellswerehomogenizedindistilledwaterandcentrifugedat550xgfor5min.Thesupernatantsolutionsweremixedwithanequalvolumeofthesamplebuffer(63mMTris-HCl,pH6.8,2%SDS,5%2-mercaptoethanol,10%glyceroland0.002%bromophenolblue)andwereheatedinaboilingwaterfor3min.Thesamples(5~50μgproteinperlane)wereelectrophoresedaccordingtothemethodofLaemmli(6)onapolyacrylamide-grADIent(8-16%)slabgel(Tefco,Tokyo)withtheelectrophoresisbuffer(25mMTris,192mMglycine,and0.1%SDS)at20mAforabout1.5hr.HogkidneyD-amino-acidoxidase(10ng,Sigma)andprestainedproteinstandards(SeeBlue,Novex,SanDiego,CA)wereruntogether.Theproteinswereelectrophoreticallytransferredfromthegeltoanitrocellulosemembrane(BA85,SchleicherandSchuell,Dasel)withtheblottingbuffer(25mMTris-HCl,192mMglycine,and20%methanol)at180mAfor1hr.

Themembranewasincubatedin5%non-fatdriedmilk(Bio-Rad)inTBS-T(20mMTris-HCl,pH7.6,137mMNaCl,and0.1%Tween-20)for1hrandthenquicklyrinsedtwicewithTBS-TandwashedinTBS-Toncefor15minthentwicefor5min.Itwasincubatedfor1hrinTBS-Tcontainingrabbitanti-hogD-amino-acidoxidaseIgG(1/3,000dilution)andquicklyrinsedtwicewithTBS-TandfurtherwashedinTBS-Toncefor15minthentwicefor5min.Themembranewasincubatedfor1hrinTBS-Tcontaininghorseradishperoxidase-labeledantibodyagainstrabbitIgGraisedinadonkey(ECLwesternblottingdetectionset,Amersham,Buckinghamshire)(1/1,000dilution).Followingthis,themembranewasquicklyrinsedthreetimeswithTBS-TandfurtherwashedinTBS-Toncefor15minthentwicefor5min.AfterdrainingexcessTBS-Tsolution,themembranewascoveredwithamixtureof2.5mleachofDetectionsolution1and2(ECLwesternblottingdetectionset)for1min.Aftertheexcesssolutionwasdrainedoff,themembranewaswrappedwithSaranWrapandexposedtoanautoradiographyfilm(Hyperfilm-ECL,Amersham)forabout5~30sec.D-Amino-acidoxidaseproteinwasdetectedasa39kDabandundertheseconditions.

PCRamplificationofD-amino-acidoxidaseCDNAfragment

TotalRNAwasextractedfromorgans,tissues,ortissueculturecellsbasicallybythemethodofChomczynskiandSacchi(7).Isogen(NipponGene,Tokyo)wasusedforthisextraction.ThefirststrandofcDNAwassynthesizedusingSuperscriptPreamplificationSystem(BRL,Gaithersburg,MD).

Asenseprimer(F:5"-GGTTAACTGAGAGGGGAGTGAA-3")andanantisenseprimer(R:5"-CCATAGTTGTGGATGACCTCTG-3")weredesignedfromthesequenceconservedincDNAsencodinghuman,mouse,rabbit,andpigD-amino-acidoxidases.TheseprimersweresynthesizedbyGreinerJapan(Tokyo).ThePCRreactionmixture(20μl)contained0.8μlofthefirststrandofcDNAsolution,0.2μMeachofthesenseandantisenseprimers,dNTPmix(0.2mMeach),0.5unitsofExpandHighFidelity(BoehringerMannheim)andthereactionbuffer.InplaceofExpandHighFidelity,LATaq(Takara,Ohtsu),AmpliTaqGold(PEAppliedBiosystems,FosterCity,CA)werealsousedsatisfactorily.Afteraninitialdenaturationat94°Cfor1min,30cyclesofPCR(denaturation:94°C,30sec;annealing:55°C,30sec;extension:72°C,45sec)wereperformedusingathermalcycler(GeneAmpPCRSystem2400,Perkin-ElmerCetus,Norwalk,CT).Afinalextensionwasdoneat72°Cfor5minandthereactionwaskeptat4°C.10μlofthereactionsolutionwereelectrophoresedon2%agarosegelsandvisualizedwithethidiumbromidestaining.ADNAfragmentofabout480base-pairswasamplifiedundertheseconditions.

Inthisprocedure,cDNAsynthesisandPCRwerecarriedoutseparately.However,thesetwostepscouldbedoneinasingletubeusingTitanOneTubeRT-PCRSystem(BoehringerMannheim)orOneStepRNAPCRKit(Takara).Bothgavesatisfactoryresults.

Northernhybridization

TotalRNAwasextractedfromorgans,tissues,ortissueculturecellsasdescribedabove.Poly(A)+RNAwaspurifiedusingoligo(dT)latexbeads(OligotexdT30Super,Takara)accordingtothemethodspecifiedbythesupplier.Poly(A)+RNAwasfinallyrecoveredintheTEbuffer(10mMTris-HCl,pH7.5and1mMEDTA).InplaceofOligotexdT30Super,theFastTrackmRNAIsolationSystem(Invitrogen,SanDiego,CA)alsoyieldedsatisfactoryresults.

Poly(A)+RNA(2~10μg)wereelectrophoresedona1.2%denaturingagarosegelaccordingtothemethodofSambrooketal.(8).Markers(0.24-9.5kbRNALadder,BRL)wereruntogether.TheRNAswerecapillary-transferredovernighttoanylonmembrane(HybondN,Amersham).Thistransfercouldbedonewithin1hrusingavacuum/pressureblotter(ModelRB-30S,NipponGenetics,Tokyo).TheRNAwasUV-crosslinkedtothemembrane(UVcrosslinker,Stratagene,LaJolla,CA).Themembranewasincubatedat65°Cfor1hrinaprehybridizationsolution[5xSSPE(1xSSPEis0.18MNaCl,10mMsodiumphosphate,and1mMEDTA,pH7.7),5xDenhardt"ssolution(8),and0.5%SDS]containing20μg/mldenatured,fragmentedsalmonspermDNA.TheD-amino-acidoxidasecDNAfragmentamplifiedabovebyPCRwaslabeledwith[-32P]dCTP(~110TBq/mmol,Amersham)basicallybythemethodofFeinbergandVogelstein(9).DNALabelingKit(Takara)orMultiprimeDNALabelingSystem(Amersham)wasused.Afterbeingheat-denatured,theprobewasaddedtotheprehybridizationsolution.Hybridizationwasdoneat65°Cforabout16hrinarollerbottleinahybridizationoven(Maxi14,Hybaid,Middlesex).Themembranewaswashedtwicefor10mineachinasolutionof2xSSPEand0.1%SDSatroomtemperature,oncefor15minat65°inasolutionof1xSSPEand0.1%SDS,andtwicefor10mineachat65°Cinasolutionof0.1xSSPEand0.1%SDS.ItwaswrappedwithSaranWrapandexposedtoanimagingplate(FujiFilm,Tokyo).Theplatewasreadinanimaginganalyzer(BAS2000II,FujiFilm).Inplaceoftheimagingplateandimaginganalyzer,conventionalautoradiographycouldbeusedtodetectthehybridizingsignal.However,thisprocessrequiredmoretime.Undertheseconditions,ahybridizing2-kbbandwasdetectedinthekidneyandcerebellumofthemouse,andinthekidney,liver,andhindbrainoftherat.

D-Amino-acidoxidaseactivitywasdetectedinthekidneysofhuman,monkey,rat,mouse,chicken,frog,carp,dace,cruciancarp,catfish,rainbowtrout,andelectricray(10,11).D-Amino-acidoxidaseproteinwasdetectedbywesternblottinginthekidneyandbrainofthemouse,andinthekidneyoftherat(12-14).RT-PCRamplifiedaD-amino-acidoxidasecDNAfragmentfromRNAextractedfromthekidneyandbrainofthemouse,andfromthekidney,liver,andcerebellumoftherat(13,14).NorthernhybridizationshowedthepresenceofmRNAforD-amino-acidoxidaseinthekidneyandcerebellumofthemouse,andinthekidney,liver,andhindbrainoftherat(13,14).

AllthevertebratesexaminedsofarhaveD-amino-acidoxidaseintheirlivers(1,2,15,16).However,themouseliverdidnotshowpositiveresultsintheD-amino-acidoxidaseenzymeactivityassay,westernblotting,RT-PCR,andnorthernhybridization.Therefore,weconcludedthatthemousedoesnothavethisenzymeinitsliver(13).Themouseisaveryuniqueanimalinthisrespect.

Northernhybridization,RT-PCR,andwesternblottinggavepositiveresultsforthepresenceofD-amino-acidoxidaseintheddY/DAO-mice(12,17).However,thekidneyandbrainhomogenatesofthesemicedidnotshowD-amino-acidoxidaseactivity(18,19).Therefore,weconcludedthattheyproducedtheD-amino-acidoxidaseproteinwithoutenzymeactivity.Thislackofactivitywasduetoasingle-basesubstitutioninthecodingregionofthecDNAforthisenzyme(17).