Eur. J. Biochem. 157, (1986) 0 FEBS 1986

Transcription

1 Eur. J. Biochem. 157, (1986) 0 FEBS 1986 Mechnism of renl peritubulr extrction of plsm glutthione The ctlytic ctivity of contrlumenl y-glutmyltrnsferse is prerequisite to the pprent peritubulr extrction of plsm glutthione Msysu INOUE, Shigeru SHINOZUKA nd Yoshims MORINO Deprtment of Biochemistry, Kummoto University Medicl School (Received December 27, 1985/Mrch 11,1986) - EJB To clrify the peritubulr mechnism for renl hndling of plsm glutthione (GSH), vrition of GSH levels in plsm, urine, kidney nd liver ws exmined fter intrvenous dministrtion of GSH to three groups of nimls; control, civicin-treted nd rts treted with buthionine sulfoximine (BSO). Tretment of nimls with BSO, potent inhibitor of de novo GSH synthesis, mrkedly reduced heptorenl GSH levels. Acivicin did not ffect these levels. Upon intrvenous injection of GSH (0.1 mmol/kg), renl GSH levels did not pprecibly chnge in ny of three niml groups. The rte of GSH disppernce from the circultion ws rpid in control nd BSO-treted rts, while it ws mrkedly retrded in nimls whose renl y-glutmyltrnsferse ws extensively inctivted by civicin. At 30 min fter dministrtion significnt mount of injected GSH ws loclized extrcellulrly (urine nd plsm) in civicin-treted nimls. By contrst, most of the GSH rpidly disppered from the extrcellulr spce in control nd BSO-treted nimls. Together with the immunocytochemicl evidence for the peritubulr y-glutmyltrnsferse [Spter, H. W., Poruchynsky, M. S., Quintn, N., Inoue, M. & Novikoff, A. B. (1982) Proc. Nut1 Acd. Sci. USA 79, the present results re fully consistent with the contention tht the ctlytic function of this enzyme is principlly responsible for the peritubulr mechnism for the renl hndling of plsm GSH. The kidney plys n importnt role in the metbolism of glutthione (GSH) nd the relted compounds in plsm with respect to interorgn trnsloction nd metbolism [l, 21. Experiments in vivo [3] nd in vitro [4] reveled tht significnt mounts of GSH come out of the liver into the blood circultion (14 nmol GSH min-' g liver-'). Recent studies, using isolted heptic sinusoidl plsm membrne vesicles, reveled tht, s in the bile cnliculr trnsport of GSH [5], the sinusoidl trnsport of GSH occurred vi crrier-medited mechnism [6]. Hhn et l. [7] showed tht rdioctivity derived from intrvenously dministered GSH ccumulted preferentilly in the kidney. Brtori et l. [8] reported tht bout 80% of GSH in the influent perfuste disppered from the medium fter single pss through the kidney. Since only bout 20% of plsm components with smll moleculr mss cn be filtered through the glomeruli by single pss, significnt prt of plsm GSH must be extrcted by non-filtrting peritubulr mechnism(s). The renl extrcting ctivity gretly exceeded the glomerulr filtrtion cpcity even when the rteril GSH concentrtion ws incresed to more thn 200 times s high s the control level [9]. There hve been severl reports on the mechnism for peritubulr hndling of plsm GSH. Anderson et l. [3] showed tht civicin, n finity-lbeling regent of glutmine midotrnsferses, decresed the frction of GSH removed Correspondence to M. Inoue, Deprtment of Biochemistry, Kummoto University Medicl School, Honjo, Kummoto-shi, Kummoto-ken, Jpn 860 Abbrevitions. GSH, reduced glutthione; GSSG, oxidized glutthione ; BSO, buthionine sulfoximine. by single pss through the kidney to vlue pproching tht filtered. Since civicin is potent inctivtor of y-glutmyltrnsferse [lo, 111, involvement of this enzyme in the peritubulr hndling of plsm GSH ws suggested. Lumenl s well s contrlumenl locliztion of the trnsferse hs recently been demonstrted histochemiclly [ nd electron microscopiclly [12]. It hs been well documented tht the trnsferse on the renl brush border membrnes rpidly hydrolyzes lumenl GSH, which ws filtered through the glomeruli or trnslocted cross the picl membrnes [15]. However, it is not known whether or not the ctlytic function of the contrlumenl y-glutmyltrnsferse is ctully responsible for the pprent extrction of peritubulr GSH. On the other hnd, using multiple-indictor dilution method with rdioctive lignds in the control nd civicintreted nimls, Rnkin nd Curthoys [I61 presented indirect evidence for GSH-trnsport system in the renl peritubulr spce. Lsh nd Jones [17] reported N+-dependent trnsport system for GSH in isolted bsolterl plsm membrne vesicles from rt. However, the pprent K, for GSH is very much higher (3 mm) thn the physiologicl concentrtion of GSH in rteril plsm (15 pm). Thus, whether or not the trnsport system in bsolterl membrnes opertes in vivo in the peritubulr extrction of GSH is not known. To clrify the pprently conflicting views on the peritubulr mechnism for hndling plsm GSH, it is importnt not only to mesure the difference in GSH level between renl rteril nd venous plsm but lso to determine its intrcellulr concentrtion. The present pper describes () plsm disppernce profiles of intrvenously dministered GSH, (b) urinry occurrence of the injected GSH, nd (c) heptorenl levels of GSH

2 606 in the control, civicin-treted nd buthionine-sulfoximine(bs0)-treted nimls. Accumultion of rdioctivity derived from [3H]glycine-lbeled GSH in vrious tissues ws lso determined. The results provided severl lines of evidence for the involvement of contrlumenl y-glutmyltrnsferse in the non-filtrting peritubulr hndling of plsm GSH. MATERIALS AND METHODS Mterils GSH, y-glutmyl-p-nitronilide, NADPH nd 5,5'-dithiobis(2-nitrobenzoic cid) were purchsed from Sigm Chemicl Co. [3H]Glycine-lbeled GSH ws obtined from New Englnd Nucler Co. Acivicin ws obtined from Dr R. H. Erhrt (The Upjohn Compny, USA). BSO, potent inhibitor of y-glutmylcysteine synthetse, ws synthesized s described previously [18]. Other regents were of nlyticl grde. In vivo experiments Plsm disppernce of the injected GSH ws mesured using mle Wistr rts, g, essentilly s described previously [19]. Under pentobrbitl nesthesi, ech niml ws intrvenously injected with 10 pmol GSH dissolved in 0.3 mi sline (ph 7.4) over period of 5 s. At indicted times fter dministrtion, blood smples were collected from the left femorl vein nd centrifuged t rev./min for 1 min in n Eppendorf centrifuge To plsm smples thus obtined were dded perchloric cid nd EDTA to give finl concentrtions of 5% nd 5 mm, respectively, nd the smples were centrifuged t rev./min for 5 min s described bove. The cid-soluble frctions were determined for glutthione. The liver nd kidney were simultneously perfused from n bdominl vein with 10 ml ice-cold 0.15 M NCl. The excised liver nd kidneys were immeditely homogenized with 6 ml nd 3 ml, respectively, ice-cold 5% perchloric cid, which contined 5 mm EDTA; this procedure effectively prevented the enzymic degrdtion of GSH during smpling. Other tissues were lso homogenized in 3 vol. cid solution, After centrifugtion of these homogentes t 4000 rev./min for 10 min, the precipittes were resuspended in 3 ml cid solution nd centrifuged gin. The combined cid-soluble frctions were nlyzed for glutthione. Urine smples were obtined by puncture of the urinry bldder nd the glutthione contents were determined. Since GSH metbolism shows diurnl rhythm [20], in vivo experiments were performed between 8.00.m. nd m. without prior fsting of the nimls. In some cses rdioctive GSH ws intrvenously injected (0.1 mmol/kg body weight) nd vrious tissues were excised nd homogenized in 3 vol. cid solution 30 min fter dministrtion. Rdioctivity in the cid-soluble frctions ws determined s described bove. Assys y-glutmyltrnsferse ctivity ws determined s described [21]. Glutthione concentrtions (GSH + 2 GSSG) of the cid-soluble frctions were determined by the recycling method bsed on GSH reductse nd 5,5'-dithiobis(2-nitrobenzoic cid) [22]. Concentrtions of oxidized glutthione in the cid-soluble frctions were determined s described previously [23]. Rdioctivity in vrious tissues ws deter- - f h CI J : - 4 0'6 T - 0 2p ' O Time ' 1' ' min) ' ' Fig. 1. Plsm clernce of intrvenously dministered GSH. Animls were treted with civicin (0.05 mmol/kg, i.v., nd 0.1 mmol/kg, s.c.) or BSO (1 mmol/kg, i.v., nd 1 mmol/kg, s.c.) 2 h before experiments. Control nimls were injected sline (0.3 ml, i.v., nd 0.3 ml, s.c.). After intrvenous dministrtion of GSH (0.1 mmol/kg), chnges in plsm concentrtion of glutthione (GSH + 2 GSSG) were determined t indicted times s described in the text. Ech point represents men f SD derived from three nimls. (0) Control nimls; (0) civicin-treted nimls; (0) BSO-treted nimls mined s described [19]. Sttisticl nlysis ws done using Student's t-test nd P vlues less thn 0.05 were considered significnt. RESULTS Plsm clernce of GSH To elucidte the mechnism by which plsm GSH is extrcted by the kidney, firely lrge dose of GSH (0.1 mmoi/kg) ws dministered intrvenously nd chnges in its plsm concentrtion were determined in control, civicintreted nd BSO-treted rts (Fig. 1). Plsm GSH decresed very rpidly in control nimls; its concentrtion reched 0.1 mm t 30 min fter the dministrtion of 10 pmol GSH/ niml. The kidney of civicin-treted nimls retined 2.4% of the trnsferse ctivity of the kidney from control untreted rts. Although the level of y-glutmyl-trnsferse is low (2.3 mu/mg protein) [24], the heptic trnsferse lso decresed in civicin-treted nimls below detectble levels (dt not shown). Consistent with the previous observtions [3], plsm GSH levels prior to loding of GSH were incresed to pm in civicin-treted nimls. The bsl level of GSH in BSO-treted rts did not significntly differ from tht in control nimls (30 pm). The rte of GSH disppernce from the circultion ws mrkedly retrded in nimls which were pretreted with civicin. After 30 min of GSH dministrtion, 1.2 mm GSH still remined in the plsm of the treted nimls. Since mjor prt of plsm GSH is extrcted by the kidney [7], the mrked retrdtion of GSH disppernce could be scribed minly to the inhibition of the renl peritubulr hndling by civicin. The rte of disppernce of the injected GSH in BSO-treted nimls ws identicl to tht in control nimls. Anlysis by the glutthione reductse method reveled tht more thn 97% of plsm glutthione ws found to be reduced form in the three niml groups.

3 Tble 1. Heptorenl glutthione levels in control, BSO-treted nd civicin-treted nimls After intrvenous dministrtion of GSH (0.1 mmol/kg), heptorenl glutthione levels (GSH + 2 GSSG) were determined. Pretretment of nimls with civicin (AT-125) nd BSO ws crried out s described in Fig. 1. Other conditions were the sme s in Fig. 1. Ech vlue represents men f SD derived from three nimls with triplicte determintions Tissue Tretment Glutthione levels fter Kidney ~ ~~ 0 min 5 min 10 min 20 min 30 min pmol/g tissue control 2.48 f f f f & 0.27 AT f f $ f f 0.36 BSO 0.68 f f f f 0.09 Liver control 5.10 f f f & f 0.47 AT f f f f 0.40 BSO 1.90 f f & f f Heptorenl GSH levels To know the metbolic fte of intrvenously dministered GSH, heptorenl GSH levels were determined in the three groups of nimls (Tble 1). Although plsm GSH levels decresed rpidly in control nimls, the renl GSH concentrtion remined constnt. The renl GSH concentrtion of civicin-treted nimls ws similr to tht of control nimls nd remined unchnged during the experiments. Since BSO is potent inhibitor of y-glutmylcysteine synthetse, renl GSH levels decresed mrkedly fter treting nimls with this inhibitor. This low concentrtion did not increse even fter GSH dministrtion. It hs been known tht the hlflife of renl GSH is bout 30 rnin [25]. Since the kidney ws homogenized with ice-cold perchloric cid solution immeditely fter excision, enzymic degrdtion of GSH during the smpling procedures should be negligible. The results indicte tht GSH in plsm ws not tken up into renl cells of these nimls in its intct form even fter injection of firely lrge dose of GSH. Heptic GSH levels were lso determined in the control, civicin-treted nd BSO-treted nimls fter intrvenous dministrtion of GSH. Heptic GSH levels remined constnt in both control nd civicin-treted rts. In contrst, the heptic GSH level of BSO-treted nimls decresed mrkedly nd this level did not increse even fter dministrtion of lrge dose of GSH. Urinry occurrence of GSH Tble 2 shows the urinry occurrence of GSH in control, civicin-treted nd BSO-treted nimls. Even when lrge dose of GSH ws given, no significnt mount of GSH ws found in urine smples of control nimls, indicting tht the kidney hs sufficient cpcity to hndle most of the plsm GSH in the renl rtery. Despite the rpid disppernce of plsm GSH nd low level of renl GSH, only trce mount of GSH ppered in urine of BSO-treted nimls. In contrst, totl of bout 6.05 p ol GSH ppered in urine in civicin-treted nimls 30 rnin fter dministrtion of 10 pmol GSH. When GSH ws not loded, the treted niml excreted 2.39 pmol GSH in urine during the sme period of 30min. Thus, frction of the totl urinry GSH, which corresponds to 3.66 ymol, could be estimted to be derived from the dministered GSH nd to hve ppered in urine in its intct form during this period. At this time the plsm GSH concentrtion of the civicin-treted nimls ws bout Tble 2. Urinry occurrence of injected GSH Urine smples were obtined from control, civicin-trcted or BSOtreted nimls (100 g body weight) 30 rnin fter intrvenous dministrtion of 10 pmol GSH nd determined for their glutthione levels (GSH + 2 GSSG) s described in the text. Pretretment of nimls with civicin nd BSO ws performed 2 h before experiments s described in Fig. 1. Other conditions were s desuibed in the text. Dt show men k SD derived from four nimls with triplicte determintions Animls GSH Urinry occurrence loded of glutthione in urine pmol Control (+) 0.01 f Acivicin-treted ( k 0.40 Acivicin-treted (+I 6.05 f 0.98 BSO-treted (+I 0.03 f mm (Fig.1). Since rts hve pproximtely 31.3 ml plsm/kg body weight, 3.76 pmol injected GSH still remined in the circultion. Thus, bout 74% of the injected GSH remined in extrcellulr comprtments (plsm nd urine) in civicin-treted nimls. Accumultion of rdioctivity in vrious tissues All tissues contin significnt mounts of GSH nd this tripeptide turns over rpidly vi interorgn metbolism [l, 2, 7, 261. To gin insight into the dynmic spects of the GSH metbolism, ccumultion of rdioctive metbolite(s) derived from [3H]glycine-lbeled GSH ws determined in vrious tissues 30 rnin fter dministrtion nd compred between the control, BSO-treted nd civicin-treted nimls. As shown in Tble 3, the mounts of rdioctivity found in liver nd kidney were rther low s compred with those found in the muscle nd urine. Thin-lyer chromtogrphic nlysis reveled tht most of rdioctivity in urine smples from control nd BSO-treted nimls cochromtogrphed with n uthentic glycine (RF = 0.45) in propn-1-ol/wter/ ceticcid(10:s:l). Thus, prt (11-13%)ofthe 3H-lbeled glycine, which ws rpidly generted from GSH, might hve escped from renl rebsorption to pper in the urine of these two niml groups. In contrst, most of rdioctivity in the urine of civicin-treted nimls ws ccounted for by intct glutthione (RF = 0.34). This is consistent with the

4 608 Tble 3. Accumultion of rdioctive metbolite(s) in vrious tissues fter intrvenous dministrtion of glutthione Under pentobrbitl nesthesi, nimls were injected with [3H]Glylbeled glutthione (0.1 mmol/kg body weight) into the right femorl vein. 30 min fter dministrtion, nimls were exsnguinted from the left femorl rtery. Liver nd kidneys were simultneously perfused from the cvl vein with 3 ml ice-cold sline solution. The excised tissues were rinsed with ice-cold sline solution nd immeditely homogenized with 3 vol. 5% perchloric cid contining 5 mm EDTA. The homogentes thus obtined were centrifuged t 750 x g for 5 min nd the superntnt frctions were removed. The precipitted frctions were resuspended with 3 vol. cid solution nd were wshed by recentrifugtion. The combined cid solution ws nlyzed for rdioctive metbolite(s). Ech vlue is expressed s the men f SD derived from four nimls. Vlues in prenthesis show the percentge of dose found in tissues. The totl weight of the muscle for 100 g rt ws ssumed to be 43 g [33] Tissues Rdioctivity control civicin BSO cpm/orgn Hert 805f f f 42 Lung (0.3) 156Of 157 (0.2) 1187k 300 (0.3) 176Of 226 Spleen (0.6) 1105f (0.7) 1093 f 81 Muscle f 1462 (0.4) f _ (25.8) (13.6) (18.3) Smll 5133f f f 139 intestine Liver (2.1) 5875 f 1237 (1.8) 6458 & (1.8) 5550f 71 Kidney (2.5) 8783f 144 (2.7) 13350f 849 (2.3) 7300 f 1131 Urine (3.7) f 4900 (5.5) f 1600 (3.0) f 1900 (11.3) (28.4) (13.2) observtions described for Tble 2, in which significnt mounts of the injected GSH ppered in urine in civicintreted nimls but not in control nd BSO-treted rts. Rdioctivity found in other tissues, such s hert, lung nd spleen, ws very low. No significnt chnge in GSH levels of these tissues ws found in three niml groups 30 min fter intrvenous dministrtion of GSH. Among vrious tissues exmined, the muscle ccumulted significnt mount of rdioctivity derived from [3H]glycine-lbeled GSH. The mount of rdioctivity ccumulted in the muscle decresed by 50% in nimls whose trnsferse ctivity ws ffinitylbeled nd inctivted by civicin. The rdioctivity found in these tissues ws 47%, 53% nd 46% of the injected dose in control, civicin-treted nd BSO-treted nimls respectively. Since rdioctivity derived from intrvenously dministered GSH ws lso loclized in tissues other thn those exmined in the present experiments [7], the residul rdioctivity might remin in the crcss. DISCUSSION The present results provide strong evidence tht the renl hndling of plsm GSH is predominntly chieved by the ctlytic function of y-glutmyltrnsferse, nd, together with the unequivocl evidence for the presence of y-glutmyltrnsferse in the renl peritubulr spce [12], fully support the suggestion [3] tht renl extrction of plsm GSH might involve non-filtering mechnism. Since it hs been suggested tht high concentrtions of civicin my non-specificlly inhibit the puttive GSH-trnsport system by lkylting the peritubulr trnslocse [16, 171, low dose (0.15 mmol/kg) of civicin ws used in the present study to minimize the possible non-specific lkyltion. Recent studies in isolted membrne vesicles [5, 6, 15, 24, 261 reveled tht heptic sinusoidl, cnliculr nd renl brush border membrnes of intct rts possess trnsport systems for GSH secretion, which were ll insensitive to civicin. N+-cotrnsport systems for mino cids nd D-glucose in renl brush border membrnes were not ffected by this inhibitor. Thus, it is likely tht civicin cted predominntly in inctivting y-glutmyltrnsferse. Although civicin is potent ffinity lbeling regent for y-glutmyltrnsferse, it is prcticlly difficult to inctivte completely the renl trnsferse in vivo. Thus, smll residul trnsferse ctivity (bout 2.4% of the control level) still remined in the kidney of civicin-treted rts. Since renl trnsferse ctivity of the intct rt kidney is extremely high (300 units/g kidney), even few percent of the residul trnsferse ctivity my be sufficient for renl ctbolism of trcer mount of 3H-lbeled GSH [16]. In the present experiments firly lrge dose of GSH (0.1 mmol/kg body weight) ws used to elicit distinct consequence of depression of y-glutmyltrnsferse ctivity. It hs been known tht trnsport cross the brush border membrnes is generlly the rte-limiting step for ctive trnstubulr secretion of nephrophilic compounds, such s p-minohippuric cid nd mercpturic cids [27, 281. Thus, fter intrvenous dministrtion of loding dose of these compounds, trnsient increse in their renl levels cn be seen [29]. It should be noted tht the hlf-life of renl GSH is significntly longer (bout 30 min) [25] thn tht of plsm GSH ( few minutes) in the intct nimls nd tht tretment of nimls with civicin mrkedly decresed the plsm clernce of the injected GSH without incresing the renl GSH levels. Tretment of nimls with civicin lso resulted in mrked glutthionemi [3]. These observtions suggest tht events occurring t the peritubulr plsm membrnes, but not the brush border membrnes, might be the rte-limiting step for the renl hndling mechnism. If ctive trnsport of intct GSH cross the renl bsolterl plsm membrnes were responsible for the rpid peritubulr hndling, significnt trnsient increse in its renl level would hve occurred fter injection of loding mount of this tripeptide. No such chnge in the renl GSH level ws observed with either the intct or civicin-treted nimls. Recent studies [17] reveled N f -dependent uptke of GSH into civicin-treted bsolterl membrne vesicles from rt kidney. However, the K, vlue for GSH trnsport ws very much higher (3 mm) thn the physiologicl concentrtion of plsm GSH (15 pm). It hs been known tht the K, vlue for GSH degrdtion by y-glutmyl-trnsferse is significntly lower (6 pm) thn the concentrtion of GSH in rteril plsm [15]. The renl bsolterl plsm membrnes contin 1 unit of y-glutmyltrnsferse/mg protein [ 171. To understnd the quntittive spect of trnsport nd degrdtion of GSH by the intct bsolterl membrne vesicles from rt kidney, severl relevnt kinetic prmeters were compred. As reflected by the rtio of the respective V,,,/Km vlues (Tble 4), the enzymic clevge of extrvesiculr GSH on the intct membrne surfce might be bout times more efficient thn the uptke of intct GSH even when the trnsport system is optimized for uptke. This suggests tht the enzymic degrdtion of peritubulr GSH might occur much fster thn the

5 609 Tble 4. Kinetic prmeters for GSH trnsport nd degrdtion by the intct renl bsolterl membrne vesicles Mechnism of V,,, Km VmrIKm GSH hndling moi min ' M min-' mg-' 1 mg protein- Trnsport 1.95 x lo-' 3.0 x x Degrdtion by the trnsferse 1.0~ 6.0~ 1.7 x lo-' These vlues were obtined from [33]. trnsport of GSH into intct tubule cells under physiologicl conditions. Consistent with this notion is the recent report by Abbot et l. [30] tht codministrtion of lbeled GSH with lrge mounts of the unlbeled constituent mino cids into the renl rtery led to lbel recoveries in the renl venous blood which re closer to those of inulin. Thus, competitive inhibition of the trnsport process for the lbeled GSH metbolites by the unlbeled mino cids occurred t the peritubulr membrnes. It should be noted tht GSH uptke by the vesicles ws effectively inhibited by probenecid, potent specific inhibitor of the renl peritubulr trnsport system for orgnic cids [27]. It hs been known tht trnsport of orgnic nions, such s p-minohippuric cid, cross the renl peritubulr membrnes is lso N+-dependent Since GSH is negtively chrged under physiologicl conditions nd the substrte specificity of the trnslocse is very brod [27], this nion trnsport system might ct in the in vitro uptke of GSH by civicin-treted bsolterl membrnes. Since the efficiency of the GSH trnsport by this system is extremely low s compred with tht of GSH degrdtion, involvement of this trnsport system in the in vivo hndling of peritubulr GSH my be unlikely under physiologicl conditions. In fct, s fr s the present results re concerned there were no dt which could be interpreted s showing the renl occurrence of significnt uptke of GSH in its intct form. Schulmn et l. [31] reported tht ptients with y-glutmyltrnsferse deficiency showed both glutthionuri nd glutthionemi. If peritubulr uptke of intct GSH were normlly operting s distinct non-filtrting mechnism, both y-glutmyltrnsferse nd the puttive peritubulr GSH trnslocse [16, 171 would be lcking in this inherited disese. This seems unlikely since such double muttion is generlly very rre. It ws reported tht, in humn subjects lso, intrvenously dministered GSH disppered rpidly from the plsm with n pprent hlf-life of 1.6 min [32]. In this cse the subjects showed cretinine clernce of 136 ml/min. If one ssume tht plsm GSH is extrcted by glomerulr filtrtion s effectively s cretinine, hlf-life of s long s 15 min would be expected for the plsm GSH. Thus, the non-filtering mechnism for GSH disppernce from the renl circultion might lso operte in humn kidney. This work ws supported in prt by grnt in id for scientific reserch from The Ministry of Eduction, Science, nd Culture of Jpn. REFERENCES 1. Curthoys, N. P. & Hughey, R. (1979) Enzyme 24, Meister, A. (1983) Science (Wsh. DC) 220, Anderson, M. E., Bridges, R. 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