AMmethyltrienoloneradioactivegradientsThegradientswerecentrifugedat200000gfor18hinanSW501rotorBeckmanInstrumentsIncPaloAltoCAFractions02mlwerecollectedusinga690densitygradientfractionat ID: 91169
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AMmethyltrienolone(radioactivegradients).Thegradientswerecentrifugedat200,000gfor18hinanSW50.1rotor(BeckmanInstru-mentsInc.,PaloAlto,CA).Fractions(0.2ml)werecollectedusinga690densitygradientfractionator(ISCO,Lincoln,NE).Boundandfreehormonewereseparatedinpostcentrifugationfractionsbythehydrox-ylapatiteprocedurethatwaspreviouslydescribed(3).Thewashedhy-droxylapatitepelletwasextractedwith1mlethanolatroomtemperaturefor30minandcentrifugedat1,500gfor5-min;thesupernatantwasdecantedintovialsformeasurementofradioactivityinaliquidscintil-lationcounter.Parallelaliquotsoftheincubatedfractionswerekept18hat4VC,andadirecthydroxylapatiteassayofboundradioactivitywasperformed(3)atthetimeofassayofthegradientfractions.In19experiments,adirectcharcoalassaywasalsoperformedafterovernightincubation;50ilofdextran-charcoalsuspensionwereaddedto0.2-mlfractions,andthetubeswereshakenat40Cfor5minandcentrifugedat5,000gfor30min.Thesupernatantwasaspiratedandassayedforradioactivity.ResultsThetechniquesforextractingtheandrogenreceptorforforeskinhavebeenadaptedfromthestudiesofFichmanetal.(11).Theandrogenreceptorsweremeasuredbysucrosedensitygradientmethodsusingradioactivegradientsaswellasadirectbindingassay.Inbothinstancesanhydroxylapatiteprocedurewasusedtoseparateboundfromfreehormone(3).Thesequentialex-tractioninbuffersoflowandhighmolarityallowedthesepa-rationoffractionsthatconsistedpredominantlyofcytosolandnuclei.ReextractionofthepelletwitheitherTEMSHbufferor500mMKClinTEMSHbufferresultedina12-15%increaseinreceptor(resultsnotshown).Becausewewishedtoassaytheamountofreceptorundervaryingconditionsofreceptoroc-cupancybyendogenoussteroidsweusedradioactivegradientsthatallowexchangebetweenreceptor-boundandfreesteroidduringtheentireperiodofcentrifugation,andinthedirectassayweincubatedthesamplesovernight(3).Undertheseconditions,itmadenodifferencewhetherthesampleswereincubatedfor1h(shorterperiodswerenotexamined)or24hbeforeapplyingthemtodensitygradients(resultsnotshown).Aspreviouslyre-portedforhumanprostate(16)andforeskin(11-13),freezingthetissueinliquidnitrogenforaslongas3dhadnoeffectonthereceptorlevels(TableI).Methyltrienolonewasusedasthebindingligandbecauseitisnotmetabolizedatasignificantratebyeithercytosolornuclearextracts(resultsnotshown)andbecauseitbindsweaklytotestosterone-bindingglobulinofplasma.Theamountofreceptorwasassayedundersaturatingconditionsofbindingligand(5nM)(Fig.1).TableI.EffectofStorageinLiquidNitrogenontheRecoveryofAndrogenReceptorAndrogenreceptorTissuecomponentFreshFrozenfmol/gtissueCytosol2024Nuclear3842Theforeskinfroma23-yr-oldmanwasdividedintotwoaliquots.Oneportion(1.8g)wasprocessedimmediately,andoneportion(3.9g)wasfrozeninliquidnitrogenfor72hbeforeprocessingasdescribedinthetext.Bindingof[3H]methyltrienolonewasassessedbythedirecthydroxylapatiteassay.mc0_6EC',Ec1IDXCYTOSOLNUCLEAREXTR-*TotalALowAffirnty|40-*Specfic30-20-/151015[3H]Methyltrienolone,nMIACTFigure1.Bindingof[31I]methyltrienolonebycytosolandnuclearex-tractsasafunctionofmethyltrienoloneconcentration.Cytosol(9.8mgprotein/ml)andnuclearextract(2.7mgprotein/ml)werepreparedfromforeskinfroma19-yr-oldman,andtheandrogenreceptorwasassessedusingthedirecthydroxylapatiteassayatvaryingconcentra-tionsof[3H]methyltrienoloneasdescribedinthetext.Hydroxylapatiteabsorptionwasusedfortheassayofthe[3H]methyltrienolone-receptorcomplexesbecauseinpreviousstudiesofthehumanprostatewehadobservedthepresenceofamethyltrienolone-bindingproteinthatisdetectedbycharcoalassaybutnotbythehydroxylapatitetechnique(16).Withthehydroxylapatitemethod,specificmethyltrienolonebindinginforeskincytosolislocalizedtothe4and9Sregionsofthedensitygradient,whereasinthehighsaltnuclearextractsitwasalwaysinthe4Sregions(Fig.2).AndrogenreceptorlevelsobtainedusingthedensitygradientassayweresimilartothoseobtainedE0._ca510152025510152025FractionNumberFigure2.Gradientdensitycentrifugationofcytosolicandnuclearex-tractsofhumanforeskinin5-20%sucrosecontaining5nM[3H]methyltrienolone.(A)Cytosol(23.4mgprotein/ml)andnuclearextract(4.7mgprotein/ml)werepreparedfromforeskinfroma6-yr-oldboy.(B)Cytosol(46mgprotein/ml)andnuclearextract(11.0mgprotein/ml)werepreparedfromforeskinfroma13-yr-oldboy.(C)Cytosol(16.7mgprotein/ml)andnuclearextract(2.9mgprotein/ml)werepreparedfromforeskinfroma23-yr-oldman.Theextractswereincubatedwith5nM[3H]methyltrienolonefor1h,and0.25-mlali-quotswerelayeredover5-20%sucrosegradientscontaining2.5gMtriamcinoloneacetonideand5nMand/or5usM[3H]methyltrienoloneandcentrifugedat250,000gfor18h.Afterfractionationofthegra-dients,eachfractionwastreatedwithhydroxylapatiteandassayedfor'Hasdescribedinthetext.AndrogenReceptorinHumanForeskin45 binding.Bindingasafunctionofagewasanalyzedastheamountofspecificmethyltrienolonebindingpergramoftissue,permilli-gramoftissueprotein,andpermilligramoftissueDNA.Thetrendsweresimilarforthethreemethods.Forcomparativepur-posesindividualvalueswereplottedpergramoftissueweightforcytosol,nuclearextract,andtotalbinding(i.e.,thesumofcytosolandnuclearbinding)(Fig.5).ThemeanvaluesplusandminusstandarddeviationsforvariousagegroupsareshowninFig.6.Theamountofreceptorincytosolinnewborninfantsaveraged-30fmol/gtissueandfellto-20fmol/gtissueby1yrofage,increasedtoamaximumof80fmol/gtissueintheteenageyears,andthendecreasedbyage30tovaluesnotsig-nificantlydifferentthaninearlyinfancy.Inthenuclearextract,CYTOSOL100rso87OG$D.'cr..E.;1:.:-I-IFigure4.Displacementof[3H]methyltrienolone(3H-R1881)bindingbyvarioussteroids.0.l-mlaliquotsofcytosol(39mgprotein/ml)andnuclearextract(8.2mgprotein/ml)fromforeskinfroma19-yr-oldmanwereincubatedfor18hat40Cwith5nM[3H]methyltri-enolonealoneorwith0.5AMmethyltrienolone(R1881),dihydrotestosterone(DHT),testosterone,3a-an-drostanediol(3a),orpro-gesterone.Bindingwasas-sessedbythedirecthydrox-ylapatiteassayasdescribedinthetext.:IT'isgoE2201801401006020100a]60°2012080I40.-00g-C.*0,.,AOsI...oAA*AALes-AAAAAIAAaNUCLLARm%NB612510203040AgeTOTAL.Figure5.Foreskinandro-*---'----genreceptorasafunctionCYTOSOLofage.Thedataareex-pressedasfemtomolesofAAspecificbindingof-,-ACTL[3H]methyltrienoloneperEXTRACTgramweightoftissueusingthedirecthydroxylapatiteassayforcytosol,nucleara.!wextract,andforthesumof506070cytosolandnuclearextract(total).receptorwasmeasurableinthenewborn,felltoalowlevelbe-tween1moandtheonsetofpuberty,increasedtoamaximumof-100fmol/goftissuebyage16-20,andthendecreasedonaverageinlaterlifetolevelsoffmol/g(Fig.6).Totalan-drogenreceptorvaluesaresimilarinpatterntothoseofthecytosolfraction.Theratioofnucleartocytosolicandrogenre-ceptorisshowninFig.6.Mostofthereceptorispresentincytosolduringchildhood.Thenuclearfractionincreasesatpu-bertyandafterage20accountsonaverageforabouthalfofthetotalreceptor.ThefindingsinnucleiareinagreementwiththoseofFichmanetal.(11)andsuggestthatphysiologicalamountsofandrogenanchorthereceptorinthenuclei.DiscussionThetechniquesutilizedinthisstudymakeitpossibletoassesstwocomponentsofandrogenreceptorinhumanforeskin-onefractionthatcanbeextractedinlowionicstrengthbuffersandanotherthatisextractedwithhighsalt.Weassume,alongwith3wU)0C,110EC;Cr104)mr_02.T-t::E-:27la_-92100.2.Z:11"C,I0crag0zcrz.p'fta,00-o',~,.!Ie.-a'jAgeGroupsFigure6.Meanlevelsofforeskinandrogenrecep-torinnuclearandcyto-solasafunctionofage.TheresultsshowninFig.5havebeengroupedasnewborn,age1-12mo,age1-5yr,6-10yr,11-15yr,16-20yr,21-30yr,31-40yr,41-50yr,andTj;&#x /R9;&#x 11.; Tf;&#x 0.6;( 0;&#x 0 1;&#x 461;&#x.88 ;ѷ.;r T;&#xm 00;50yr.ThedataareexpressedasmeansplusorminustheSEofthemean.Thenumberofsamplesareshowninparenthe-ses.Inthebottompaneltheratioofmeannu-cleartomeancyto-plasmicreceptorhasbeenplottedforeachagegroup.46C.G.Roehrborn,J.L.Lange,F.WGeorge,andJ.D.Wilson1tuc150V-120_90H60InF308Eo604020.'._mExNUCLEAREXTRACT100-6040K20..eI.- (orcentrifugationingradientscontaining[3H]methyltrienolone)followedbyadsorptionoftheandrogen-receptorcomplextohy-droxylapatitecircumventstheproblemofcontaminationoftis-sueswithtestosterone-bindingglobulinfromplasmaandmin-imizesthechanceofmeasuringmethyltrienolone-bindingpro-teinsthatmaybereceptordegradationproducts(16).Withthetechniquesusedinthepresentstudywehaveob-serveda