Impairment of mineralocorticoid receptor (MR)-dependent biological response by oxidative stress and aging

Impai men o Mine aloco icoid Recep o (MR)-dependen Biological Response by Oxida i e S ess and Aging CORRELATION WITH POST-TRANSLATIONAL MODIFICATION OF MR AND DECREASED ADP-RIBOSYLATABLE LEVEL OF ELONGATION FACTOR 2 IN KIDNEY CELLS* Recei ed o publica ion, Oc obe 2, 2001 and in e ised o m, Decembe 18, 2001 Published, JBC Pape s in P ess, Janua y 23, 2002, DOI 10.1074/jbc.M109530200 G aciela Piwien-Pilipuk‡, An onio Ayala§, Albe o Machado§, and Ma io D. Galigniana‡¶ F om he ‡Depa amen o de Quı´mica Biolo´gica, Facul ad de Ciencias Exac as y Na u ales, Uni e sidad de Buenos Ai es, Ciudad Uni e si a ia, 1428 Buenos Ai es, A gen ina and he §Depa amen o de Bioquı´mica, B oma ologı´a y Toxicologı´a, Facul ad de Fa macia, Uni e sidad de Se illa, 41012 Se illa, Spain Acu e and ch onic ea men s o mice wi h he glu a- hione-deple ing agen , L-bu hionine-(SR)-sul oximine (BSO), impai ed he mine aloco icoid ecep o (MR)- dependen biological esponse by inhibi ing aldos e - one binding. This s e oid-binding inhibi ion was ully e e sed when educing agen s we e added o kidney cy osol ob ained om mice ea ed o 5 h, bu i was only pa ially e e sed in cy osol ob ained om mice ea ed o 10 days. Al hough he oligome ic s uc u e o he MR-hsp90 he e ocomplex was always una ec ed, a dec eased amoun o MR p o ein was e idenced a e he long e m ea men . Such a dele e ious e ec was co ela ed wi h a pos - ansla ional modi ica ion o MR, as demons a ed by an inc eased le el o ecep o ca - bonyla ion. In addi ion, a ailu e a he elonga ion/ e - mina ion s ep was also obse ed du ing he ecep o ansla ion p ocess in a e iculocy e lysa e sys em. Thus, a high poly ibosomes/monome s a io and bo h inc eased p o eolysis and dec eased ADP- ibosyla able concen a ion o elonga ion ac o 2 (EF-2) we e shown. Impo an ly, simila obse a ions we e also pe o med in i o a e deple ion o glu a hione. No wi hs anding he EF-2 unc ional dis up ion, no all enal p o eins we e equally a ec ed as he MR. In e es ingly, bo h EF-2 and MR exp essed in old mice we e simila ly a - ec ed as in L-bu hionine-(SR)-sul oximine- ea ed young mice. We he e o e p opose ha a d ama ic de- ple ion o glu a hione in kidney cells mimics he cumu- la i e e ec o aging which, a he end, may lead o a enal mine aloco icoid dys unc ion. The biological e ec s o aldos e one (ALDO) 1 a e media ed by he mine aloco icoid ecep o (MR), a ligand-dependen ansc ip ion ac o ha belongs o he s e oid ecep o class o nuclea ecep o s. The ansc ip ional ac i a ion o he MR in epi helial cells igge s a se ies o e en s ha a e esponsible o he egula ion o he in e nal medium, i.e. Na ⫹ and H 2 O e en ion and K ⫹ and H ⫹ elimina ion. S e oid ecep o s exis as nuclea o cy oplasmic he e ocom- plexes associa ed o he 90-kDa hea shock p o ein (hsp90) chape one sys em (1, 2). Rega dless o i s subcellula localiza- ion, his associa ion s abilizes he ecep o in i s ho mone binding and ansc ip ionally inac i e o m. I is hough ha upon ligand binding, he s e oid ecep o s unde go a con o ma- ional change ha leads o he dissocia ion o he hsp90-he - e ocomplex, dephospho yla ion, dime iza ion, ansloca ion in o he nucleus ( o cy oplasmic ecep o s), hype phospho y- la ion, and binding o speci ic ho mone- esponsi e elemen s. None heless, he ac ual empo al sequence o his cascade o e en s emains unclea . In con adic ion o wha was p e i- ously hough , i has been shown ha he dissocia ion o he hsp90-he e ocomplex upon s e oid binding is no necessa ily he i s s ep in he signaling pa hway (3, 4). Consis en wi h his obse a ion, i has also been pos ula ed ha he ecep o - hsp90 complex equi es in ac cy oskele al acks o mo e e i- cien ly owa d he nucleus (5). Fo his ansloca ion p ocess, he ac i i y o he Se /Th phospha ases seems o play a key ole (3, 6, 7), and cy oplasmic dynein may be he mo o p o ein equi ed o mo e he complex on he ilamen s (8, 9). In e es ingly, he MR nuclea ansloca ion is ab oga ed by oxida i e s ess in in ac kidney cells (2). Se e al s udies (10– 13) pe o med in i o wi h bo h cell- ee sys ems and cells in cul u e ha e demons a ed ha cys eine g oups on he MR play an essen ial ole in s e oid binding. We ha e ecen ly p o ided di ec e idence (14) ha he MR unc ion is also a ec ed in i o in a simila manne as shown in i o. Thus, inhibi ion o ALDO binding o enal MR was obse ed a e adminis e ing mice a ansi ion s a e inhibi o o ␥ -glu amyl- cys eine syn he ase, L-bu hionine-(SR)-sul oximine (BSO). The consequen GSH de iciency pa alleled he inhibi ion o he mine aloco icoid biological esponses o he same ex en a e bo h sho (5 h) and long (5 days) pe iods o ea men . We demons a ed ha his e ec was due o he inhibi ion o he s e oid binding o MR due o oxida ion o essen ial cys eine g oups a he han changes on he ecep o p o ein concen a- ion. Howe e , we subsequen ly obse ed ha he le el o ex- p ession o enal MR is sys ema ically dec eased a e GSH deple ion o longe imes (i.e. 10 days). Inasmuch as a wide a ie y o enal diso de s in ol e he o e p oduc ion o eac i e oxygen species (15–17), a dec eased le el o MR exp ession may be ele an o unde s and he egula ion o he c i ical acid * This wo k was suppo ed by g an s om Consejo Nacional de In- es igaciones Cien ı´ icas y Te´cnicas de la Repu´blica A gen ina and Minis e io de Sanidad y Consumo de Espan˜a G an FIS 96-1442. The cos s o publica ion o his a icle we e de ayed in pa by he paymen o page cha ges. This a icle mus he e o e be he eby ma ked “ad e - isemen ” in acco dance wi h 18 U.S.C. Sec ion 1734 solely o indica e his ac . ¶To whom co espondence should be add essed: 1301 Medical Sci- ence Resea ch Bldg. III, Dep . o Pha macology, he Uni e si y o Mich- igan Medical School, Ann A bo , MI 48109. Tel.: 734-764-5414; Fax: 734-763-4450; E-mail: [email p o ec ed]. 1 The abb e ia ions used a e: ALDO, aldos e one; MR, mine aloco - icoid ecep o ; hsp90, 90-kDa hea shock p o ein; BSO, L- bu hionine- (SR)-sul oximine; CH, cumene hyd ope oxide; GSH, educed glu a hi- one; GME, glu a hione monoe hyl es e ; MDA, malondialdehyde; EF-2, elonga ion ac o 2; P , o al pep ides; Pn, nascen pep ides; DTT, di- hio h ei ol; MOPS, 4-mo pholinep opanesul onic acid; SGK, se um- and glucoco icoid-dependen kinase. THE JOURNAL OF BIOLOGICAL CHEMISTRY Vol. 277, No. 14, Issue o Ap il 5, pp. 11896–11903, 2002 © 2002 by The Ame ican Socie y o Biochemis y and Molecula Biology, Inc. P in ed in U.S.A. This pape is a ailable on line a h p://www.jbc.o g11896 This is an Open Access a icle unde he CC BY license. base and elec oly e balance unde no mal o pa hological ci - cums ances (e.g. oxida i e s ess, me al in oxica ions, aging, e c.). A p o ound imbalance be ween oxidan s and an ioxidan s has been ela ed o enal diso de s, om minimal change ne- ph i ic synd ome o obs uc i e neph opa hy (16, 18). Mo e- o e , pa ien s wi h end-s age enal ailu e on main enance hemodialysis a e ch onically exposed o he oxida i e s ess gene a ed by eac i e oxygen species due o pe dialy ic neu o- phil ac i a ion (19). Inc eased concen a ion o malondialde- hyde (MDA) (20), an in e media e p oduc gene a ed by lipid pe oxida ion, and dec eased le els o GSH (15) we e also ound in plasma and e y h ocy es o hemodialyzed pa ien s. Because he MR is no an expo ed p o ein, a dec eased a e o p o ein syn hesis and/o an inc eased p o ein deg ada ion in enal cells may explain he obse ed diminu ion o MR le els unde ch onic oxida i e condi ions. The p oposi ion ha oxy- gen adical-media ed oxida ion is a ma king s ep in p o ein u no e is suppo ed by se e al obse a ions ( e iewed in Re s. 21 and 22 and e e ences he ein) so ha mos o he s udies we e mainly ocused on p o ein deg ada ion. In ag ee- men wi h his no ion, i has been demons a ed ecen ly ha he u no e o oxidized p o eins is enhanced in ib oblas s ia p o eosomal ac i i y (23). On he o he hand, he molecula mechanism o p o ein syn hesis ailu e du ing he onse o oxida i e s ess has no been ex ensi ely s udied and, conse- quen ly, is no comple ely de ined. Because o his ac and also because oxidized MR seems o be mo e s able o he mal deg- ada ion (al hough incapable o bind s e oid) han i s “ educed” o m (10), we decided o analyze he e ec o oxida i e s ess gene a ed by GSH deple ion on he indi idual s eps o polypep- ide syn hesis in kidney. By using he GSH-deple ing agen BSO as an expe imen al model, we aimed o s udy he exp es- sion o enal MR, and we co ela ed his p ocess wi h he elonga ion phase o p o ein syn hesis unde in i o and in i o condi ions. Because he elonga ion phase only equi es a small numbe o ac o s as compa ed wi h he ini ia ion s ep, we also ocused ou analysis on he MR ansla ion p ocess. In pa ic- ula , we sea ched o pu a i e pos - ansla ional modi ica ions o elonga ion ac o 2 (EF-2), he main p o ein implica ed in he elonga ion s ep. Finally, we discuss ou molecula indings om he pe spec i e o an in eg a ed p ocess ha may ake place unde no mal and pa hological condi ions. MATERIALS AND METHODS Reagen s—[1,2- 3 H]ALDO (50 Ci/mmol), [U-adenosine- 14 C]NAD (850 mCi/mmol), NaB[ 3 H] 4 (63 Ci/mmol), [ 35 S]me hionine (1,000 Ci/mmol), L-[3,4- 3 H] aline (55 Ci/mmol), and 125 I-conjuga ed coun e an ibodies we e om Pe kinElme Li e Sciences. Reduced GSH, GSH monoe hyl es e (GME), L-bu hionine-(SR)-sul oximine (BSO), cumene hyd ope - oxide (CH), p o ein A-Sepha ose, bu yla ed hyd oxyanisole, ␣ - ocoph- e ol, de e oxamine, manni ol, cys eine, sodium asco ba e, ca alase om bo ine li e , and diph he ia oxin om Co ynebac e ium diph he iae we e om Sigma. RU486 was a kind gi om Roussell-Ucla (Romain- ille, F ance). Comple e-Mini TM p o ease inhibi o mix u e was om Roche Molecula Biochemicals. DNA encoding o ull-leng h human MR was gene ously p o ided by D . R. E ans (24). TNT Quick-coupled T ansc ip ion-T ansla ion ki was om P omega Co p. (Madison, WI). Rabbi polyclonal an ibodies agains EF-2 (25) and he MR (26) we e desc ibed p e iously. The mouse monoclonal IgG an ibody agains hsp90 was pu chased om S essGen (Vic o ia, B i ish Columbia, Can- ada). Donkey an i- abbi and goa an i-mouse IgG-ho se adish pe oxi- dase an ibodies we e om Pie ce. Chemiluminescence eagen s we e pu chased om Ame sham Biosciences. Deple ion o GSH—BSO was dissol ed in e hanol/p opylene glycol/ saline solu ion (3:5:32). Two daily in amuscula injec ions o 2.5 mmol BSO/kg we e gi en o BALB/c mice (see below) a in e als o 12 h (8:00 and 20:00 h) du ing he indica ed imes. In co- ea men s wi h GME, h ee doses o 2.0 mmol/kg o he es e we e also injec ed (8:00, 14:00, and 20:00 h). The ALDO-dependen sodium e en ion and po assium elimina ion we e measu ed in u ine samples collec ed as desc ibed p e iously (14, 27). S e oid Binding Assays—Thi y-day-old and 18-mon h-old male BALB/c mice we e ad enalec omized by do sal app oach and main- ained wi h Pu ina Die 1, saline solu ion supplemen ed wi h 50 ␮ g/ml dexame hasone, and eshwa e ad libi um. Two days be o e sac i ice, he s e oid was omi ed om he saline solu ion, and ood was emo ed he p e ious nigh . Kidneys we e excised a e ex ensi e pe usion wi h ice-cold phospha e saline solu ion, and enal co ex-medulla in e - phases we e homogenized in 1 olume o bu e MOPS/Mo (20 mM MOPS, 5 mMEDTA, 2 mMEGTA, 10% glyce ol, 2 mMDTT, 20 mM Na 2 MO 4 , a pH 7.5) con aining 1 able o p o ease inhibi o mix u e pe 5 ml o solu ion. Homogena es we e cen i uged a 67,000 ⫻g o 45 min a 3 °C, and he supe na an o his cen i uga ion was e e ed o as cy osol. Renal cy osol was incuba ed o 12 h a 0 °C wi h 20 nM [ 3 H]ALDO and 1.0 ␮ MRU486 o mask he sligh c oss- eac ion o ALDO wi h he glucoco icoid ecep o . A 500- old excess o adioine ALDO was used o de e mine he nonspeci ic binding (20% o he o al). Bound s e oid was sepa a ed om ee s e oid by adding 1 olume o 2% cha coal, 0.2% dex an 15–20. ALDO binding o kidney MR was meas- u ed in i o as desc ibed be o e (14, 28). B ie ly, 30-day-old male mice we e ea ed wi h ehicle, BSO, o BSO and GME o he indica ed pe iods. An in ape i oneal injec ion o 10 ␮ Ci o [ 3 H]ALDO and 20 ␮ g o RU486 (⫾30 ␮ g ALDO) was gi en. Kidneys we e emo ed a e 20 min and homogenized in bu e MOPS/Mo lacking DTT. F ee [ 3 H]ALDO was clea ed om cy osol by adso p ion wi h cha coal/dex an, and he samples we e di ided in o equal ac ions. The speci ic binding was measu ed ei he wi hou u he ea men o a e eincuba ing cy- osol wi h 10 nM[ 3 H]ALDO (⫾500- old ALDO) o 3ha 0°Cin he absence o p esence o 2 mMDTT. F ee ace was adso bed wi h cha coal, and he adioac i i y was measu ed. Renal Poly ibosomal P o iles—Poly ibosomes and monome ic ibo- somes we e analyzed in pos -mi ochond ial supe na an s ea ed wi h deoxychola e (⬃65% o he o al enal RNA) by using a 20–40% linea suc ose g adien as desc ibed p e iously (25). The RNA p o iles we e moni o ed by measu ing he abso bance a 260 nm and hen semi- quan i ied by planime y. Inco po a ion o [ 3 H]Valine in o To al and Nascen Polypep ide Chains—The p ocedu e o he de e mina ion o o al p o eins was simila o he one desc ibed p e iously (25) o li e . Mice we e anes- he ized wi h pen oba bi al (50 mg/kg); he abdomen was opened, and 25 ␮ Ci o L-[3,4- 3 H] aline in 200 ␮ l o saline solu ion was injec ed in he enal a e y. Tissue samples we e aken a he indica ed imes and immedia ely ozen unde liquid ni ogen un il homogeniza ion in 3 olumes o 0.3 Msuc ose. Unde hese expe imen al condi ions, he adioac i i y was de ec ed in kidney wi hin 5–10 s a e he injec ion o adioac i e aline and con inued a an app oxima ely linea a e o 2.5–3.0 min. The e o e, we began o ob ain issue samples 20 s a e he injec ion and du ing a o al pe iod o 2 min. Kidney homogena es in 0.3 Msuc ose we e dilu ed wi h 1 olume o a bu e con aining 100 mMT is and2mMmagnesium ace a e and cen i uged o 15 min a 4,000 ⫻g. Two ml o he supe na an we e mixed wi h 0.2 ml o 20% sodium deoxychola e, incuba ed on ice o 30 min, and hen cen i uged a 8,000 ⫻g o 15 min. P o eins in he esul an supe na an we e used o quan i y o al and nascen chains. To measu e adioac i i y o o al enal p o ein, an aliquo o his supe na an was ea ed wi h 10% ichlo oace ic acid a 90 °C o 1.5 h. P ecipi a ed p o eins we e edis- sol ed wi h p o osol, and he adioac i i y was coun ed. The inco po a- ion o labeled aline in o nascen polypep ides was de e mined laye - ing 1.5 ml o supe na an on 6 ml o a medium con aining 1 Msuc ose, 1mMmagnesium ace a e, and 1 ml o a 100,000 ⫻gsupe na an ob ained by 1 h o ul acen i uga ion o kidney homogena e (1:2) in 50 mM ie hanolamine, 5 mMMgCl 2 ,25mMKCl and 0.25 Msuc ose (pH 7.3). The mix u e was cen i uged a 25,000 ⫻g, and he esul an pelle was esuspended in 1 ml o dis illed wa e . P o eins we e p ecip- i a ed and he adioac i i y coun ed as desc ibed abo e. Immunop ecipi a ion o MR—We ollowed a s anda d echnique as desc ibed p e iously (3, 14). B ie ly, enal cy osol was incuba ed wi h he abbi an i-MR immune se um (o non-immune abbi se um) p e- bound o p o ein A-Sepha ose and washed ou imes wi h ice-cold MOPS bu e supplemen ed wi h 100 mMNaCl and 0.01% Nonide P-40, and MR and hsp90 we e iden i ied by Wes e n blo analysis. In Vi o T ansc ip ion and T ansla ion o he MR—The p ocedu e was pe o med using he TNT Quick-coupled T ansc ip ion/T ansla ion ki om P omega Co p. (Madison, WI) acco ding o he manu ac u e ’s ins uc ions. Recombinan phMR3750 DNA encoding o ull-leng h human MR (24) inse ed in o polylinke EcoRI si e o pGEM4 (P omega Bio ech) was used as a empla e o ansc ip ion wi h T7 polyme ase MR Inac i a ion by Oxida i e S ess 11897 ollowed by ansla ion in he p esence o [ 35 S]me hionine. P o eins we e esol ed by SDS-PAGE and au o adiog aphed. When he incuba- ions we e pe o med in he p esence o oxidan s, he e iculocy e lysa e was p eincuba ed o 60 min a 25 °C wi h ei he 2.0 mMCH and/o 5.0 mMBSO. In he p o ec ion assays agains oxidan s, a educing solu ion was also simul aneously added o he medium a he ollowing inal concen a ions: 20 ␮ Mbu yla ed hyd oxyanisole, 200 ␮ M ␣ - ocophe ol, 5 mMGSH, 5 mMcys eine, 2 mMDTT, 1 mMsodium asco ba e, 20 mM manni ol, 3 mMde e oxamine, and 0.5 mg/ml ca alase. ADP- ibosyla ion o EF-2—ADP- ibosyla ion was pe o med by a modi ica ion o he me hod used by Galicka e al. (29). Renal EF-2 was immunop ecipi a ed om enal cy osol wi h he abbi polyclonal an i- body aised agains EF-2 p ecoupled o p o ein A-Sepha ose. The im- mune pelle was washed wice wi h MOPS bu e con aining 100 mM NaCl and 0.01% Nonide P-40 and wice wi h 20 mMT is bu e a pH 7.4. The pelle s we e incuba ed o 1ha 37°C wi h 50 ␮ l o a solu ion con aining 20 mMT is, 10 mMDTT, 10 ␮ M[ 14 C]NAD, and 5 ␮ go diph he ia oxin. The pelle s we e washed ou imes wi h 1 ml o 20 mM T is bu e , and he adioac i i y inco po a ed o he immunopu i ied EF-2 was coun ed. Pu i ica ion o EF-2—EF-2 was isola ed om he ibosome- ee ex- ac o abbi e iculocy e lysa e as desc ibed by Ryazano and Da y- do a (30). The pu i y o he inal p epa a ion was e i ied by SDS- PAGE ollowed by Coomassie Blue G-250 s aining and pa allel Wes e n blo ing wi h an i-EF-2 an ibody. The a e o inco po a ion o [ 35 S]Me o hMR in abbi e iculocy e lysa e sys em supplemen ed wi h pu e EF-2 was measu ed a e alkaline hyd olysis ollowed by ichlo oace ic acid p ecipi a ion as desc ibed p e iously (31). Miscellaneous—Renal GSH concen a ion was quan i ied by an en- zyma ic assay as desc ibed p e iously (14). Ca bonyl con en in ei he enal mic osomes o immunopu i ied p o eins was measu ed by educ- ion wi h i ia ed sodium bo ohyd ide acco ding o Lenz e al. (32). MDA was measu ed wi h he hioba bi u ic s anda d me hod desc ibed by Es ebaue and Cheeseman (33). S a is ical es s we e ca ied ou by analysis o a iance ollowed by Bon e oni analysis. RESULTS E ec o GSH Deple ion on he Mine aloco icoid Response— Table I shows ha he enal concen a ion o GSH was d a- ma ically dec eased when mice we e ea ed wi h he ␥ -glu- amylcys eine syn he ase inhibi o , BSO. Such an e icien deple ion was achie ed as soon as 5 h a e a single injec ion o BSO. On he o he hand, a co- ea men wi h he cell-pe me- able es e GME ully p e en ed he dele e ious e ec o BSO by p ese ing (and e en inc easing) he in acellula le els o GSH. In ag eemen wi h he onse o an oxida i e in acellula milieu, an inc eased concen a ion o bo h MDA, a p oduc gene a ed by he b eak down o hyd ope oxides, and p o ein ca bonyls we e also measu ed a e acu e (5 h) and p olonged (3 and 10 days) ea men . On he o he hand, he le els o hese wo ma ke s o oxida ion we e indis inguishable om un ea ed con ols when mice we e co- ea ed wi h BSO and GME. To e alua e he in i o mine aloco icoid e ec , a sa u a ing dose (28) o 2 ␮ g o ALDO pe 100 g o body weigh was injec ed, and he an i-na iu e ic and kaliu e ic e ec s we e measu ed. ALDO dec eased he Na ⫹ /K ⫹ u ina y a io by 80% in con ol ad enalec omized mice. Howe e , he biological esponse o ALDO was a enua ed a e 5ho BSOinjec ion, so a 2.5- old highe Na ⫹ /K ⫹ a io was measu ed. This e ec was mo e sig- ni ican o longe pe iods o ea men (3 and 10 days). Taken oge he , hese esul s clea ly con i m (14) ha he deple ion o enal GSH impai s he mine aloco icoid biological esponse. In e es ingly, he da a shown in Table I also sugges a simila e ec o aging on he mine aloco icoid esponse. Thus, 18- mon h-old animals also exhibi high le els o p o ein ca bonyls and MDA, as well as a dec eased biological esponse o ALDO as compa ed wi h 30-day-old animals. In Vi o [ 3 H]ALDO Binding Assay—To e alua e he MR bind- ing capaci y in i o, con ol and BSO- ea ed mice we e in- jec ed wi h [ 3 H]ALDO, and kidneys we e excised a e 20 min, a ime when enal adioac i i y eaches a maximum le el in kidney (28). Renal cy osol was ob ained in MOPS/Mo bu e wi hou DTT; ee ace was clea ed by adso p ion wi h cha - coal/dex an, and he samples we e di ided in o equal ac- ions. The speci ic binding (Fig. 1) was measu ed ei he wi hou u he ea men o a e a eincuba ion o cy osols wi h [ 3 H]ALDO in he absence o p esence o 2 mMDTT. As ex- pec ed, he ea men wi h BSO dec eased he s e oid binding capaci y o MR unde in i o condi ions, whe eas his inhibi- ion was ully p e en ed by co- ea men wi h GME. When adiolabeled cy osols we e eincuba ed in i o wi h [ 3 H]ALDO, he speci ic binding emained unchanged (g ay ba s), indica - ing ha he in i o ALDO labeling had sa u a ed he MR binding capaci y. Consis en wi h he no ion ha an oxida i e milieu inhibi s he s e oid binding capaci y o MR by oxida ion o essen ial cys eine g oups, eincuba ion o hese cy osols in he p esence o DTT eco e ed he speci ic binding o cy osolic MR ob ained om BSO- ea ed mice (black ba s). Impo an ly, such eco e ing was o al in mice ea ed o 5 h, bu i was only pa ial in cy osols ob ained om mice ea ed wi h BSO o 3 and 10 days. Thus, ⬃20 and 35% o he MR s e oid binding capaci y was no eco e ed, espec i ely. Again, old mice ex- TABLE I Oxida i e s ess ab oga es he mine aloco icoid biological e ec Ad enalec omized 30-day-old o 18-mon h-old mice we e ea ed wi h BSO o BSO and GME o he indica ed imes. Con ols we e ea ed wi h ehicle only. The mine aloco icoid e ec (Na ⫹ /K ⫹ a io) was measu ed in i o a e injec ing a sa u a ing dose o ALDO as desc ibed unde “Ma e ials and Me hods.”Con en s o GSH in enal cy osol and MDA and CO we e also quan i ied in mic osomal ac ions. Resul s a e he mean ⫾ S.E. (n⫽6). Condi ion Renal GSH a MDA b CO b Na ⫹ /K ⫹ a io c 30-day-old mice 2.51 ⫾0.21 16.1 ⫾2.9 351.3 ⫾33.1 0.17 ⫾0.04 BSO 5 h 0.34 ⫾0.15 d 37.5 ⫾9.3 e 544.4 ⫾22.7 d 0.44 ⫾0.10 d 3 days 0.18 ⫾0.06 d 40.1 ⫾1.8 d 635.9 ⫾21.9 d 0.50 ⫾0.11 d 10 days 0.15 ⫾0.08 d 61.4 ⫾1.9 d 729.6 ⫾45.0 d 0.58 ⫾0.05 d BSO ⫹GME 5 h 2.75 ⫾0.14 18.1 ⫾3.0 370.7 ⫾20.8 0.15 ⫾0.02 3 days 4.04 ⫾0.61 e 14.4 ⫾1.2 318.0 ⫾13.1 0.19 ⫾0.09 10 days 4.62 ⫾1.01 e 13.7 ⫾3.1 304.1 ⫾21.2 0.21 ⫾0.12 18-Mon h-old mice 1.87 ⫾0.45 e 36.0 ⫾4.2 d 608.3 ⫾32.1 d 0.37 ⫾0.03 d a Values a e gi en in ␮ mol/g o enal issue. b Values a e gi en in mmol/mg o mic osomal p o ein. c The Na ⫹ /K ⫹ a io in con ol mice was 1.27 ⫾0.14. d Values di e en om con ol (30-day-old mice) a p⬍0.001. e Values di e en om con ol a p⬍0.010. MR Inac i a ion by Oxida i e S ess11898 hibi ed simila p ope ies as hose shown by long e m BSO- ea ed young mice. Associa ion o MR wi h hsp90—Because he associa ion o hsp90 wi h MR is an absolu e equi emen o bind s e oid, we analyzed he co-immunopu i ica ion o hsp90 wi h enal MR a e ea men wi h BSO. The ba g aph in Fig. 2 depic s he ALDO binding capaci y measu ed in i o in cy osols ob ained om con ol and ea ed mice, because hese expe imen al esul s a e simila o hose shown in Fig. 1 unde in i o condi ions. Mo eo e , a cons an hsp90/MR op ical densi y a- io (0.19 ⫾0.02) was ob ained o all he Wes e n blo s shown a he op o Fig. 2 a e a densi ome ic scanning. These co-immunop ecipi a ion assays demons a e ha oxida i e s ess does no dec ease he MR binding capaci y by dis up ing he associa ion o MR wi h he chape one complex. Howe e , he amoun o MR (and hence, hsp90) eco e ed in he he e o- complex a e 10 days o ea men wi h BSO (condi ion 5) was one- hi d lowe han he amoun o MR p o ein eco e ed om ei he con ol cy osol (condi ion 2), cy osols om BSO- ea ed mice (condi ions 3 and 4), o GME- and BSO- ea ed mice (condi ion 6). These esul s demons a e ha he dec eased s e oid binding capaci y o MR measu ed in enal issue was due o a lowe concen a ion o MR a he han an oxida i e dis up ion o he MR-hsp90 in e ac ion. Wes e n blo analysis o o al cy osol esol ed by SDS-PAGE (shown below he ba g aph) e idenced ha he cy osolic hsp90 concen a ion was g ea ly inc eased due o he onse o oxida i e s ess, and such induc ion was ully p e en ed by co- ea men wi h GME (con- di ion 6). A Wes e n blo o ␤ - ubulin was also pe o med in he same samples, and no change was e idenced o his essen- ial cy oskele al p o ein. Impo an ly, Fig. 2 also demons a es ha all he ea u es obse ed in ch onically s essed young mice we e also p esen in un ea ed old mice (compa e condi ions 5 and 7), ein o cing he simila i ies poin ed ou be o e be ween oxida i e s ess and na u al aging. In Vi o T ansc ip ion and T ansla ion o Human MR—The dec eased concen a ion o MR ob ained by ch onic oxida i e s ess can be due o ecep o deg ada ion o a less e icien ecep o syn hesis. The e a e e idences in a o o bo h possi- bili ies. Thus, i is known ha adically media ed damaged p o eins a e o en unc ionally inac i e, and hei un olding was associa ed wi h enhanced suscep ibili y o p o eases (22). Howe e , we ha e epo ed p e iously (10) ha he oxidized MR p o ein, al hough incapable o bind s e oid, seems o be qui e s able as compa ed wi h i s educed coun e pa . Mo e- o e , ecen e idence suppo s he no ion ha p o ein syn he- sis may decline unde oxida i e condi ions by changes in he polypep ide elonga ion a e (34, 35). The e o e, we ocused ou s udy on he a ious s eps o p o ein syn hesis unde oxida i e condi ions. We i s analyzed he exp ession o a DNA empla e encoding o human MR by using he abbi e iculocy e in i o an- sc ip ion/ ansla ion sys em. A main 35 S-labeled p oduc can be seen as a 110-kDa band in Fig. 3A, as his molecula weigh is compa ible wi h he size o human MR (24). Impo an ly, his 110-kDa band was also e ealed by Wes e n blo wi h he an i-MR an ibody (da a no shown). P eincuba ion o e iculo- cy e lysa e wi h BSO ailed in a ec ing he MR ansla ion in a signi ican o m (Fig. 3A, compa e lane 2 e sus 1). In pa , his ailu e may be due o he mechanism o ac ion o he ansi ion s a e enzyma ic inhibi o . In e ec , BSO is p esen in a pos -mi ochond ial medium ha lacks an e icien sou ce o pe oxide adical p oduc s gene a ed du ing, o example, an ac i e oxida i e me abolism. Then he simple inhibi ion o he GSH syn hesis unde hese in i o condi ions may no be su icien o a ec signi ican ly he ansla ion machine y. Tha his may be he case is suppo ed by he signi ican inhibi ion o MR exp ession achie ed wi h cumene hyd ope - oxide (CH) (Fig. 3A,lane 3), a known gene a o o eac i e oxygen in e media es (25, 36, 37), and also because o he po en ia ion ob ained when bo h agen s, BSO and CH, we e used oge he (Fig. 3A,lane 5). As expec ed o a adically media ed e ec , he CH-dependen inhibi ion o MR syn hesis was abolished when he lysa e was p eincuba ed wi h CH in he p esence o a educing mix u e (Fig. 3A,lane 4) con aining 20 ␮ Mbu yla ed hyd oxyanisole, 200 ␮ M ␣ - ocophe ol, 5 mM GSH, 5 mMcys eine, 2 mMDTT, 1 mMsodium asco ba e, 20 mM manni ol, 3 mMde e oxamine, and 0.5 mg/ml ca alase. Inas- much as he syn hesis o MR was pe o med in i o, wo s eps a e hen equi ed o comple e he p ocess, empla e ansc ip- ion ollowed by ansla ion. I could be possible ha a de- c eased p oduc ion o MR may be due o a ailu e in he o me p ocess a he han in he ansla ion s ep. The e o e, we pe - FIG.1.In i o ALDO binding o kidney MR. Thi y-day-old male mice we e ea ed wi h ehicle (con ol), BSO, o BSO and GME o he imes indica ed below he g aphs. Then an in ape i oneal injec ion o 10 ␮ Ci o [ 3 H]ALDO and 20 ␮ g o RU486 (⫾30 ␮ g ALDO) was gi en. Kidneys we e emo ed a e 20 min and homogenized in a bu e lack- ing DTT. F ee [ 3 H]ALDO was clea ed by adso p ion wi h cha coal/ dex an, and he samples we e di ided in o equal ac ions. The speci ic binding was measu ed ei he wi hou u he ea men (whi e ba s)o a e a eincuba ion o he cy osol o 3ha 0°C wi h 10 nM[ 3 H]ALDO (⫾500- old ALDO) in he absence (g ay ba s) o p esence (black ba s)o 2m MDTT. The ALDO binding capaci y measu ed in i o in 18-mon h- old mice is also shown (old mice). Resul s ep esen he means ⫾S.E. o ou animals pe g oup. Di e en om con ols a *,p⬍0.001; **, p⬍ 0.005; and ***, p⬍0.010. FIG.2. Co-immunop ecipi a ion o hsp90 wi h he MR. Mice we e ea ed wi h BSO alone o co- ea ed wi h BSO and GME o he indica ed imes. Kidney cy osol was hen ob ained, and he MR was immunop ecipi a ed. A Wes e n blo o MR and co-immunop ecipi- a ed hsp90 is shown a he op o he igu e. An aliquo o his cy osol was used o pe o m a s e oid binding assay (ba g aph) and depic ed as he means ⫾S.E. (n⫽4). A second aliquo o cy osol was Wes e n- blo ed o hsp90 and ␤ - ubulin (shown on he bo om o he g aphic). Condi ions a e as ollows. 1, Non-immune pelle was ob ained by incu- ba ion o con ol cy osol wi h a p eimmune abbi IgG an ibody. 2, Con ol cy osol was om un ea ed mice. Cy osol om BSO- ea ed mice o 5h(3), 3 days (4), and 10 days (5). 6, Cy osol was om mice co- ea ed wi h BSO and GME o 10 days. 7, Cy osol was om 18- mon h-old mice. As compa ed wi h condi ion 2, di e ences o speci ic binding a e signi ican a p⬍0.001 o condi ions 3–5, and p⬍0.005 o condi ion 7. MR Inac i a ion by Oxida i e S ess 11899 o med di ec s udies on he ini ia ion and comple ion s eps o he polypep ide syn hesis by analyzing he poly ibosomal p o- iles in e iculocy e lysa es unde no mal and oxida i e condi- ions. A ep esen a i e linea suc ose g adien is depic ed in Fig. 3B. I shows ha an inc eased ibosomal s a e o agg ega- ion was ob ained unde oxida i e condi ions. Thus, he poly- somes/monome s a io was 4- old highe in ea ed lysa es han in con ol samples (66 e sus 16, espec i ely). The highe polysomes/monome s a io obse ed in CH- ea ed lysa e is he consequence o bo h a 2- old inc eased amoun o polysomes and also a 50% educ ion o he single 80 S ibosome o ms. I is known ha he cycling be ween single ibosomal subuni s ( o ms no in ol ed in ansla ion) and poly ibosomes is e y apid (38, 39), and hose ibosomes eleased a chain e mina- ion may ei he become monome ic ibosomes o may be con- e ed in o na i e subuni s (31, 40) unless he ini ia ion is supp essed, in which case accumula ion o single ibosomes occu s (41–43). The e o e, he esul s shown in Fig. 3Bag ee wi h he no ion ha he elonga ion/ e mina ion s ep should be mo e a ec ed by oxida i e s ess han he ini ia ion s ep du - ing he ansla ion p ocess. The main p o ein implica ed in he elonga ion s ep is he EF-2. The e o e, we analyzed he EF-2 le el by Wes e n blo - ing aliquo s o e iculocy e lysa e incuba ed wi h BSO and CH, and no di e ences we e obse ed o he o al concen a ion o EF-2 in any condi ion (da a no shown). I is known ha ADP- ibosyla ion o a peculia diph hamide esidue p esen on EF-2 abolishes i s abili y o ansloca e he pep idyl- RNA om he A-si e o he P-si e on he ibosome (44). Fu he mo e, ADP- ibosyla ion o EF-2 wi h diph he ia oxin and NAD ha e been used as an indica o o he ac i e EF-2 ac ion (25, 29, 34, 45). The e o e, we immunopu i ied EF-2 om e iculocy e lysa e and de e mined he amoun o ac i e p o ein. Fig. 3Cdemon- s a es ha he amoun o ADP- ibosyla able EF-2 was e- duced by 50 and 80% in CH- and CH/BSO- ea ed samples (condi ions 3 and 5, espec i ely). This educed le el o ac i e EF-2 was no obse ed when he incuba ion was pe o med in he simul aneous p esence o he educing mix u e. Taken oge he , he esul s shown in Fig. 3 sugges ha i is en i ely possible ha he dec eased MR concen a ion obse ed in GSH-deple ed mice may lie on he inabili y o enal cells o achie e e icien ly he comple ion o he nascen p o ein, mo e speci ically due o ans o ma ion o EF-2. The Addi ion o Pu i ied EF-2 Reco e s he T ansc ip ion o hMR in Vi o—Gi en he numbe o p o eins ha a e likely o be modi ied du ing oxida i e s ess, a di ec ole o EF-2 canno be ensu ed om ou p e ious expe imen s. None heless, i he damage o EF-2 is one o he easons o he obse ed dec ease in hMR le els du ing he ansc ip ion, he addi ion o pu i ied EF-2 o he ansc ip ion/ ansla ion sys em should co ec such de iciency. The e o e, we i s pu i ied EF-2 om abbi e iculocy e lysa e and adjus ed he amoun o be added o he ansla ion medium by compa ison wi h he endogenous le el o EF-2 p esen in e iculocy e lysa e. The Wes e n blo shown in Fig. 4Ashows ha 0.3 ␮ l o pu i ied EF-2 ( e e ed o as 1⫻) ma ches he concen a ion o endogenous EF-2 in 5.0 ␮ lo e iculocy e lysa e, so ha we used his ela i e amoun o EF-2 as a e e ence. We hen supplemen ed he CH- ea ed e iculocy e lysa e employed as a ansc ip ion/ ansla ion sys- em wi h pu i ied EF-2. The hMR ansla ion p oduc s a e shown in Fig. 4B. As can be seen, he inhibi o y e ec o oxida i e s ess on he hMR ansla ion was p e en ed when pu i ied EF-2 was added o he incuba ion medium (Fig. 4B, compa e lane 3 e sus lane 2). On he o he hand, he addi ion o pu i ied EF-2 p eincuba ed wi h 2 mMCH exhibi ed no e ec on he le el o exp ession o hMR (Fig. 4B, compa e lanes 4 e sus lane 1). Fig. 4Cdepic s he a es o syn hesis o hMR measu ed as inco po a ion o [ 35 S]me hionine o he acid-insoluble ac ion. The a e o syn hesis o 35 S-labeled hMR was g ea ly dec eased unde oxida i e condi ions, whe eas he addi ion o pu i ied EF-2 o he medium was able o co ec he ansla ion p ocess in a concen a ion-dependen manne . In con as , when he ansla ion mix u e was supplemen ed wi h he same p epa- a ion o EF-2 ha had been p einac i a ed wi h CH, he co ec ion o he hMR ansla ion was no obse ed. The pu i- ied EF-2 p o ein emains s able unde he condi ions used o he p eincuba ion wi h CH (as judged by SDS-PAGE ollowed by Coomassie Blue s aining), al hough i s ADP- ibosyla ion by diph he ia oxin was ully abolished (da a no shown). Taken oge he , hese esul s clea ly indica e ha EF-2 may be esponsible o he obse ed dec ease o hMR le el du ing he ansla ion p ocess unde oxida i e condi ions. Renal Poly ibosomal P o iles—In iew o he p e ious obse - a ions, we nex analyzed he polysome p o iles in kidney cy- osol a e mice we e ea ed wi h BSO. Resul s a e shown in Fig. 5. Consis en wi h he abo e-desc ibed in i o e ec , Fig. 5Ashows an inc eased ibosomal s a e o agg ega ion when oxida i e s ess was gene a ed in i o by ea men wi h BSO o 3 and 10 days (25 and 82% la ge poly ibosome peak, FIG.3.In i o ansla ion o human MR. A, elec opho e ic analysis o he ansla ed p oduc . The ansc ip ion/ ansla ion sys em was p e ea ed o 30 min a 25 °C as ollows: lane 1, un ea ed con ol; lane 2,5m MBSO; lane 3,2mMCH; lane 4,2mMCH and he educing mix u e desc ibed unde “Ma e ials and Me hods”;lane 5,5m MBSO and 2 mMCH. The ansla ion eac ion was pe o med in he p esence o [ 35 S]me hionine. P o eins we e esol ed by SDS-PAGEand au o adiog aphed. The a ow shows he band o MR when a Wes e n blo e ealed i . B, suc ose g adien o abbi e iculocy e ibosomes. Poly ibosomal p o iles om no mal lysa e (solid line) and CH- ea ed lysa e (do ed line) we e de e mined as desc ibed unde “Ma e ials and Me hods.”The a ow shows he 80 S monome s. C, ADP- ibosyla able concen a ion o EF-2. EF-2 was immunop ecipi a ed om e iculocy e lysa e p e ea ed in he same condi ions as desc ibed o A(lanes 1–5). A Wes e n blo o his immunop ecipi a ion is shown a he op o he ba g aph o each condi ion. The ac i e concen a ion o EF-2 was measu ed by using a s anda d eac ion wi h [ 14 C]NAD and diph he ia oxin. The ba g aph ep esen s he means ⫾S.E. (n⫽3) o he pe cen age o ac i e EF-2 wi h espec o he un ea ed lysa e (condi ion 1) s anda dized as 100% (11.3 ⫾1.9 pmol/mg). Condi ions 3 and 5a e signi ican ly di e en om condi ion 1 a p⬍0.001. MR Inac i a ion by Oxida i e S ess11900 espec i ely). On he o he hand, he poly ibosomal p o ile o BSO- and GME-co- ea ed mice was indis inguishable om ha ob ained wi h un ea ed mice (Fig. 5B). In addi ion, he poly ibosomes/monome s a io was also inc eased in 18-mon h- old mice (116%), s eng hening he no ion ha aging is a p oc- ess whe e cumula i e damage by oxida ion a ec s he p o ein syn hesis machine y. We hen measu ed he polypep ide chain comple ion ime. This pa ame e is accep ed as a quan i a i e exp ession o he a e o pep ide chain elonga ion and e mina ion (25, 34, 46, 47). The adioac i i y inco po a ed in o bo h nascen pep ides in poly ibosomes (Pn) and o al pep ides (P ) was plo ed agains he ime a e he injec ion o [ 3 H] aline in o he enal a e y. The alue o Pn o each animal was hen di ided by he co esponding alue o P , and he Pn/P a io was inally plo ed agains he ime (Fig. 5C). I i is assumed ha he adioac i e amino acid will mee he ibosomes in he middle o he ansla ion o a mRNA o a e age size (25, 46, 47), so he whole pep ide on he ibosome should be labeled when a ull cycle is comple ed. In u n, hose chains ha ha e been e mi- na ed and eleased will be only 50% labeled. A e a second cycle, 1 ull uni will inc ease he pool o eleased pep ides, whe eas he Pn/P a io should ha e been educed o 25%. Thus, he ime equi ed o educe Pn/P om 50 o 25% should be ep esen a i e o he a e age comple ion ime (elonga ion and e mina ion s eps). This ime is independen o bo h he numbe o ibosomes engaged in he p ocess and a ia ions in he ini ia ion s ep and can be calcula ed di ec ly om he slope o he unc ion. In ou hands, he a e age comple ion ime inc eased om 58 o 98 s in mice ea ed wi h BSO o 10 days. Old mice e idenced a comple ion ime equal o 158 s. Damage o Renal MR and EF-2—We hen measu ed he ADP- ibosyla able con en o EF-2 immunopu i ied om kid- ney cy osol o BSO- ea ed mice. Fig. 6 shows ha he ac i e amoun o EF-2 was educed nea ly 50% a e 10 days o GSH deple ion (ha ched ba s). In u n, he p o ein ca bonyl con en in he immune pelle s was inc eased 2- old (black ba s). Bo h dele e ious e ec s we e o ally p e en ed by GME. On he o he hand, he con en o ca bonyls measu ed in MR immu- nopu i ied om BSO- ea ed mice (whi e ba s) was inc eased 40% wi h espec o un ea ed animals. A simila le el o MR ca bonyla ion was also measu ed in 18-mon h-old mice. In he expe imen shown in Fig. 2, we demons a ed ha he qua e na y s uc u e o he MR-hsp90 he e ocomplex was no a ec ed by oxida i e s ess. Those Wes e n blo s did no show lowe molecula mass p o eoly ic agmen s o MR. This obse - a ion ag ees wi h he esul s shown in Fig. 3Aand hose desc ibed p e iously (10). On he o he hand, Pa ado e al. (34) ha e ecen ly epo ed ha a li e EF-2 does unde go ag- men a ion upon he onse o oxida i e s ess induced by CH. As a consequence, we s udied he pu a i e agmen a ion o cy o- solic EF-2 in mouse kidney a e ea men wi h BSO. Fig. 6B shows he p o ein p o ile ob ained wi h he an i-EF-2 an ibody in enal cy osol. Thi y-day-old un ea ed mice exhibi ed a main band o EF-2 a he expec ed molecula mass o 100-kDa (Fig. 6B,lane 1). Besides his band, cy osol om BSO- ea ed mice also exhibi ed a majo p o eoly ic agmen a 39 kDa and wo mino bands a 51 and 67 kDa (lanes 2 and 3). The ull p e en ion o he EF-2 agmen a ion obse ed in cy osol o GME co- ea ed mice (lane 4) p o es ha p o eolysis o EF-2 in i o depends on he onse o oxida i e s ess. Thus, old mice also exhibi a agmen a ion pa e n o EF-2 simila o ha gene a ed by he deple ion o GSH in young mice (lane 5). Howe e , he o al amoun o ull-leng h EF-2 was also signi - ican ly dec eased in old mice, an obse a ion ha is consis en wi h he signi ican ly slowe hal - ansi ime o nascen polypep ides obse ed in Fig. 5C o his g oup o animals. Diminished EF-2 le els we e also ound in li e o CH- ea ed a s (34). Cu iously, despi e he lowe ull-leng h EF-2 le el obse ed o old mice, Fig. 6Balso shows ha he amoun o p o eoly ic agmen s emained unchanged. This obse a ion may be ela ed o he inhibi ion o he p o eosome ac i i y desc ibed in ce ain old cells (23), which in u n leads o he cyclic accumula ion o damaged and agg ega ed p o eins. DISCUSSION In his wo k we demons a ed ha oxida i e s ess impai s he mine aloco icoid biological esponse by wo di e en mo- lecula mechanisms. As suppo ed by he expe imen s de- sc ibed in Table I and Figs. 1 and 2, one o hese inhibi o y mechanisms in ol es he pos - ansla ional modi ica ion o he ecep o p o ein by oxida ion. Consis en wi h he esul s e- po ed in he li e a u e (10–14), i is mos likely ha oxidiza- ion o essen ial cys eine g oups is he main esponsibili y o such inhibi ion. In he sho ime (hou s), he inhibi o y e ec obse ed by deple ion o GSH can be ully e e sed in i o by incuba ing enal cy osol wi h DTT o can be o ally p e en ed in i o by co-adminis e ing GME. A second ha m ul mecha- nism able o a ec he mine aloco icoid esponse was e i- denced a e se e al days o GSH deple ion, and i a ec ed he p o ein ansla ion sys em a he elonga ion/ e mina ion s eps. Because poly ibosomes can be o med in i o (Fig. 3B) and in i o (Fig. 5, Aand B), and he e was a dec eased numbe a he han accumula ion o monome s, i is unlikely ha he ini ia ion s ep can be as s ongly a ec ed as he comple ion s ep (41–43). In ag eemen wi h he no ion ha he elonga ion/ e mina ion s ep is a ec ed by he onse o oxida i e s ess, he a e o ansla ion in i o was es o ed a e addi ion o pu i- ied EF-2 o he incuba ion medium (Fig. 4). Mo eo e , he hal - ansi a e o nascen polypep ide chains in enal cells is almos wice as slow in BSO- ea ed mice as in he un ea ed con ols. The obse ed e ec s on p o ein syn hesis du ing he BSO-dependen deple ion o enal GSH a e simila o hose FIG.4. The addi ion o pu i ied EF-2 o e iculocy e lysa e p e en s he ha m ul e ec o a low edox po en ial medium on he ansla ion p ocess. A, ela i e amoun o pu i ied EF-2. Va ious amoun s (0.3, 0.6, and 1.2 ␮ l) o he inal p epa a ion o pu i ied EF-2 we e compa ed by Wes e n blo ing wi h he endogenous le el o EF-2 p esen in 5 ␮ l o e iculocy e lysa e. The a io 0.3 ␮ l o pu e EF-2 o 5 ␮ l o e iculocy e lysa e is e e ed o as 1 ime. B, ansla ion o hMR in e iculocy e lysa e sys em. The ansla ion eac ion was pe o med as desc ibed o Fig. 3Band au o adiog aphed o he [ 35 S]Me -labeled hMR. Condi ions a e as ollows: 1, un ea ed con ol; 2, lysa e ea ed wi h 2 mMCH; 3, lysa e ea ed wi h 2 mMCH and supplemen ed wi h 2 imes pu i ied EF-2; 4, lysa e ea ed as in condi ion 3 bu EF-2 was incuba ed o 1ha 25°Cwi h2m MCH. C, a e o syn hesis. The adioac i i y associa ed o he ichlo oace ic acid-insoluble p oduc was measu ed as a unc ion o he ansla ion ime. Condi ions a e as ollows: un ea ed con ol (solid ci cles), lysa e ea ed wi h 2 mMCH (open ci cles), lysa e ea ed wi h CH and supplemen ed wi h EF-2 as ollows: 1⫻(open squa e), 2⫻(solid squa es,solid line), o 2⫻s o ed o 24 h in a bu e con aining 2 mMCH (solid squa es,do ed line). Resul s a e he a e age o wo independen expe imen s pe o med by duplica e. MR Inac i a ion by Oxida i e S ess 11901 desc ibed in he li e o a s ea ed wi h he adical dono cumene hyd ope oxide (25, 34). We emphasize ha we we e unable o gene a e an e icien oxida i e s ess wi h BSO in adul a s and olde mice. BSO is a compound ha seems o be e icien o induce oxida i e s ess only on ce ain animal mod- els such as guinea pigs, newbo n a s, o young mice (48). Ou esul s p o ide clea e idence ha he impai men o he mine aloco icoid biological esponse is co ela ed wi h an inc eased con en o issue MDA, a high ca bonyla ion le el o MR and EF-2, and a dec eased amoun o biologically ac i e EF-2 (as de e mined by i s low ADP- ibosyla able le el). In e - es ingly, no all p o eins a e a ec ed by oxida i e s ess o an equi alen ex en . Thus, he concen a ion o hsp90 was g ea ly inc eased, whe eas enal ␤ - ubulin emained un- changed a e he ea men wi h BSO (Fig. 2). In a p e ious wo k (14), we ha e also analyzed he ac i i y o se e al p o eins ela ed o he mechanism o ac ion o MR, such as ci a e syn hase, 11 ␤ -hyd oxys e oid dehyd ogenase, Na ⫹ /H ⫹ an i- po , and Na ⫹ /K ⫹ -ATPase. The e we e no d ama ic changes in any o hese p o eins. Mo eo e , he Na ⫹ /K ⫹ -ATPase ac i i y seems o be p ese ed due o an inc eased numbe o ac i e pumps, so ha a dec eased speci ic ac i i y o he Na ⫹ /K ⫹ - ATPase pumps was in e ed om hese obse a ions. Oxida- i e s ess is in insically associa ed wi h a s a e o inc eased u no e o biomolecules induced by ele a ed a es o eac i e oxygen species. Howe e , ex ensi e s udies ha e ecen ly dem- ons a ed ha o he p o eins also inc ease hei ac i i y upon oxida i e s ess in a signi ican manne , o example c-Jun NH 2 - e minal kinase (49), p70-S6 kinase (50), Ak /PKB (51), glucose-6-phospha e dehyd ogenase (52), PDK1 (53), and SGK (54), among many o he examples. In e es ingly, he las wo p o eins a e ela ed o he mine aloco icoid biological e- sponse. Thus, he se um- and glucoco icoid-dependen kinase (SGK) has been linked o he ALDO-dependen mechanism o ac i a ion o he epi helial sodium channel (55, 56). In u n, he e exis s subs an ial e idence showing ha SGK is egu- la ed by he phosphoinosi ide-dependen kinase 1, PDK1. The e o e, i seems ha he e also exis se e al egula o y mechanisms ha a e simul aneously igge ed by oxida i e s ess, so ha such a compensa o y egula ion a enua es dam- aging e ec s like hose e idenced he e o he MR-media ed biological esponse and he ansla ion machine y. On he o he hand, aging is ela ed o a dec ease in he s ess esponse and he loss o a low edox po en ial milieu (22, 57). I has been epo ed ecen ly (25) ha oxida i e s ess induced by xenobio ics dec ease p o ein syn hesis, as his e ec is due o a dec eased e iciency in he elonga ion s ep du ing he ansla- ion p ocess. Simila conclusions we e eached when aging- ela ed e ec s we e also compa ed wi h ee adical damage (34). Because he ALDO binding capaci y o enal MR de- FIG.5.Poly ibosomal p o iles and polypep ide comple ion ime in kidney. A suc ose g adien o enal polysomes was pe o med a e 30-day-old mice we e ea ed unde he ollowing condi ions (polysomes/monome s a ios a e gi en in pa en heses). A, un ea ed con ols (solid line) (13); mice ea ed wi h BSO o 3 days (dashed line) (40), o 10 days (do ed line) (59). B, un ea ed 30-day-old con ols (solid line) (13), un ea ed 18-mon h-old mice (dashed line) (47); 30-day-old mice co- ea ed wi h GME and BSO o 10 days (do ed line) (12). C,[ 3 H] aline inco po a ed in o enal Pn and P was measu ed in 30-day-old mice ea ed o 10 days wi h ei he ehicle (black ci cles) o BSO (whi e ci cles). The Pn/P a io o un ea ed 18-mon h-old mice is also shown (black squa es). The comple ion ime was calcula ed om he slope o each unc ion. The poin s ep esen he means ⫾S.E. o ou mice. FIG.6.Oxida i e damage o enal MR and EF-2. A, p o ein ca bonyla ion and ADP- ibosyla ion o immunopu i ied EF-2. Thi y-day-old mice we e ea ed o 10 days wi h BSO o GME and BSO. The ca bonyl g oup con en o MR and EF-2 and he ADP- ibosyla able le el we e measu ed in immunopu i ied samples. The same pa ame e s we e also measu ed in immune pelle s ob ained om un ea ed 18-mon h-old mice. Ba s ep esen he means ⫾S.E. (n⫽4) o ADP- ibosyla able EF-2 con en (ha ched ba s), ca bonyl g oups in EF-2 (black ba s), and he MR (whi e ba s). Resul s a e p esen ed as a pe cen age o he alue measu ed in un ea ed 30-day-old animals (23.5 ⫾1.8 nmol o MDA/mg p o ein, and 387 ⫾40 nmol and p o ein ca bonyls g oup/mg p o ein). Values a e signi ican ly di e en a *, p⬍0.005, and **, p⬍0.010. B, p o eoly ic agmen a ion o enal EF-2. P o eins om enal cy osol we e esol ed by SDS-PAGE, and EF-2 was isualized by Wes e n blo ing. Condi ions a e as ollows: 1, cy osol om 30-day-old mice; 2, cy osol om BSO- ea ed mice o 3 days; 3, cy osol om BSO- ea ed mice o 10 days; 4, cy osol om mice co- ea ed wi h BSO and GME o 10 days; 5, cy osol o un ea ed 18-mon h-old mice. A ows on he igh side show molecula weigh ma ke s. A ows on he le side show he ull-leng h EF-2 band and i s main deg ada ion p oduc s a 39, 51, and 67 kDa. MR Inac i a ion by Oxida i e S ess11902 c eases wi h aging, and because he simila pa e n e idenced in his wo k be ween he ha m ul e ec s o GSH deple ion and aging, we may ce ainly specula e ha he cumula i e damage gene a ed along he li e ime by oxida i e s ess may a ec enal cells (and o he issues as well) in a simila manne as ha desc ibed he e o mice ea ed wi h BSO. None heless, a key conund um such as whe he o no p o ein damaging is p ima y o seconda y in aging s ill emains o be answe ed. I is easonable o s a e ha he ex en o any ype o oxida- i e s ess mus be exace ba ed by a dec eased e iciency in he na u al an ioxidan compounds. In es iga ion o he GSH en- zyma ic sys em in pa ien s wi h ch onic enal ailu e has e- ealed ha he ac i i ies o GSH pe oxidase and GSH educ- ase as well as he plasma GSH concen a ion we e signi ican ly educed (15). Consis en wi h he p edominan syn hesis o GSH pe oxidase in he enal ubule, a d ama ic dec ease in plasma enzyma ic ac i i y was also obse ed. De- ple ion o GSH can also con ibu e o enal dys unc ion because his hiol is no only an e icien adical sca enge bu is also an impo an de oxican o elimina ing di e en elec ophilic compounds o exogenous and endogenous o igin ia glu a hione S- ans e ase ac i i y. The e o e, GSH de iciency may con ib- u e o he accumula ion o ha m ul compounds. In summa y, he molecula indings p esen ed in his wo k con ibu e o an eme ging pic u e ha shows a dec eased enal MR-dependen biological esponse as a consequence o he de- ple ion o he GSH an ioxidan sys em. 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