Volume 309, numtmr 3, 303-305 FEB$11520 © 1992Federation o1"Europt'an Bioeh¢micul S~icti,~ 0014S793~91~$.00

,~pteml~r 1992

Endothelin I hydrolysis by rat kidney membranes T o m Yamaguchi, Masanki Fukase, M a k o t o Arao. Toshitsugu Sugimoto and K a z u o Chihara TMrd Divi~io,. Depart, lent of Medicine. Nobe Untv¢tstt)~ School of Medicine. l¢obe, dapat~

Received 8 July 1992; revi~:l version received 15 July 1992 HydrolyJi,= ol'¢ndoth=lil~ l by rat kidney membran~ ~ats invcJtiilated min~ a r¢'~r,~-phz~ HPLC and an autonutted I~s.pham protein r e . q a ~ r . F_.ndothelin I w~,= hydrolyz~;d into four major fragments which w=m d=t~:ted by HPL¢, Pho.phoramidon, an inhibitor of neutral endo~ptid~t,c 24,1 l, almost =ompl=t~y supp~_~d_ the production of thr¢~ fralrnrntr,, but on= fralr,mcnt was not affcrt~ by tl'~ inhibitor, Anallais ofN-terminal ~quence.s of the d¢l~radation products revcal~ that the pho~phoramidon-~cmlitiv¢ fralmenta were s~=rated by cl¢~vai~ at th~ Seal-Lea" bond of endoth¢lin I that wa~ ideation| with it~ clcavall~ ~it¢ by purified rat ¢ndoi~ptida~ 24,1 I, reported previously. Th= phozphoramidon.in~miti,,~ frallrnent was produc~.d by cl~val~ at ~u~=Asp '', which was distinct from the sites by endopeptida~ 24,11, but ¢orRtponded to that by a phosphoramidon.in~ndtivc m©tallo-endop~ptidar,~ recently it,elated from rat kidney mcmbran¢~ by us [(1992) Eur. J. Bioch~m, 204, 547-552]. Kinetic determination o1" endothclin I hydroly~lis by the i ~ l a t ~ eaaym¢ yielded walu~ of ,~,,=71.5 a M and ka,.= 1.49 s"~' Siv[n~ a ratio of A'.,/E,~=2,O8 x tO' tC',M", The/f~ value wutl much hisher and th¢ L'.d/~. value wa,~ much lower than tho~t £or rat ~ndo~ptida~ 24, I 1 reportad previou,,ly, Thus, endop.ptidarm 24,11 app*ar,, to h>,drolyz~ ¢ndoth~lin I more ¢fficicntly than the iml~t=d enzyme dot~. Both enzymes may play phyr,iologir.al rol~ in the rn¢taboli~m ofendothelin I by rat kidney m~ntbrun~ in vivo, Endotlzclin 1', Kidney mgmbran¢, M¢tallo-cndopeptldas¢; Endopeptida~ 24,1 i: Pcptid¢ hydrol~is; Rat kidney

I. INTRODUCTION Recent eviden~ has revealed the importance of kid. hey m¢mbran= mctallo-¢ndopeptidases in the metabo-

lism of biologically active peptides, Neutral endo~pti. dase 24,11 is one of the well-characterized renal enzymes and is known to participate in the metabolism of some peptide hormones [1.2]. For instance, this enzym~ efficiently hydrolyzcs atrial natriuretic peptide (ANP) in vitro [3-5], and the physiological importance of en. dopeptidas¢ ~,4,11 in ANP metabolism has bccn further shown by in rive studies in animals and man [6-8] in which administration of specific inhibitors of the en. zyme caused increases in circulating ANP concentration with a concomitant aulFnentation in its physiological actions such as diuresis and natriuresis. In contrast, very little is known about the metabolism of endotbelin 1, a 21-amino acid peptide with a powerful uasoconstricting activity [9]. Several recent studies, h o w l e r , suBs~.st that renal endopeptidase 24,11 is also involved in the metabolism of endothelin l [10-12]. Membrane fractions from kidney homogenates con. tained a potent hydrolytic activity for cndoth¢lin 1 which was strongly inhibited by phosphoramidon, an inhibitor of endopeptidas¢ 24,11 [10]. Purified rat and bovine endopeptidase 24, i I were shown to cleave endo. thelin 1 initially at the gets--Lea6 bond, followed by Correspondenceaddress:T. Yarnasuchi, Third Division, Departraent of Mcdicia¢, Koho Univ~rsiq¢ $¢hool of Medicine, K o ~ 650, .Iapan. Fv~: (81) (78) 371 6468,

Published by Eh'¢vier $denee Pubtldter=B, V.

cleavage at AsptU-II¢ t', in a l~shion inactivating the peptide [11,12]. As there have bc¢n few studies on the m~ahanisms involved in endoth=lin 1 degradation by kidney membranes, however, it is still unclear whether endopeptidar¢ 24,11 is the only degrading ermyrrm integrated in kidney membranes to act on the metabolism ofendothelin 1 molecules, Indeed, a metallo-cndopeptid a ~ re~ntly purified to homogeneity from rat kidrmy membranes by us [13] has the ability to hydrolyz¢ various peptid= hormones such as endoth=lin I. ANP and parathyroid hormone in vitro [14], and seems to have the potential to metabolize rndothelin I and other biologicallyactive peptides in kidneys in vivo. The enzyme hydrolyzed =ndothelin 1 in a limited fashion at the Leu~7-As#" bond in vitro [14], which was clearly distinct from the sites cleaved by cndopeptida~¢ 24,11. In addition to the cleavage site of endotbelin 1, this isolated metallo-enzym¢ was also distinguished from endopeptidas¢ 24.11 by being iasensitive to phosphoramidon and in having an oli$om=tric structure with a mol~ular weight of the native form of 250 kDa and of its subunits of 88 kDa [13]. In this work, to further clarify the mechanism ofendoth=lin I degradation by kidney membranes, we analysed the degraded fragments using a reverse-phase HPLC and an automated ~s.phas¢ protein sequencer. and found that besides endolmptidas¢ 24.11 a new enzyme, isolated by us from the kidney membrane fraction [!3,!~, was .~nvo!ve-din th~ _..¢~_ho_~.m of'¢n~_ot~tia I. Both enzymes may be physiolo$i~lly important in the renal metabolism of endoth¢lin ! in rive. 303

Volume 309. number 3 2. M A T E R I A L S

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2.1. Muteriuh' Endothelin I, phosphommidon and umastatin were obtained from the Peptid¢ Institute, O~aka, Japan, All other chemicals were of the hillhesl Ilrude commercially ~vailable,

;

A

2.2, Pr~pututi., . f t,t khtn~.¢ ,~u~br,,~s Rut kidney membranes were prepared according to the method o( Stephenson and Kcnny [15], 2.3. Hydr.b'~i~ . f e,duthdi, I and pep!idc.seque,rv unulj's¢~ 2 0 / J M endothclin I. ~t P8 ~¢ kidney m~mbranes and lifo pM amustatin, an inactivator ofaminolx:ptida=, w¢re di~olved in 20 mM Tris-HCi buffer, pH 8,0, in a total volume of 2~0 #l, Sample= were

incubated at 37*C for 16 h and rea=tions were terminated by add!nil t .'2,5/JI 100% (by vol,} a¢ctic acid. The mixt ares were then fractionated by HPLC on an ODS 120T rcvcraz.phase column (C,=: 4.5 x 250 ram: Toyo Soda, Tokyo, Japan) whh a linear ilradient of0,-45~ a~tonit rile in 0,lql, trifluoroacetic acid f,~r 40 rain at a flow mt¢ of I mt/min, Elation was monitored by absorbante at 215 am, Dell~dation prod. acts were collected in tc=t tul~s and freeze.dried. The amino acid =quences of them ~mmpl~ were determined with an Applied Biosys. terns model 4"/0A ilat.ph:t= ~qucncer and model 120A HPLC an.l),zcr ~ystem.

I

I

I

I

I

I

0

10

20

30

40

50

,L

i .J

B

,m

|

2.4. Ki, eti¢ d¢lermiuuti#, for emlulh¢li, I ILrdr.l.rxix bj' u metallu.

e~ulupeptitl,se isultttcd frm, r¢H kithte.r mrmbrtttwx Kinetic,, for ¢ndothelin I hydrolysis by a m=mllo,cndol~=ptida~ rat,truly isolated from rat kidney mcmbran¢~ [I 3] were measured by quamitativ¢ HPLC analy=biof reaction mixtures. The it,cuberS.ontv.i,~tare (volume 250/Jl) ¢onlained 2.1"/nM (411ng) rat enzyme and 5-1~0 /JM endothelin 1 in 20 mM Tris.HCI buffer, pH 8.0.. Sampl~ were incubated at 37"C for 2 h and the re.actions wcrc stopped by adding 12.$ pl 100"& {by voi,) acetic acid. The samples were titan analy'~d by HPLC us d©~ril~d above, Product i:~.ak ar¢~s were converted to concentration by delia!as the arem, m~sured after complete hydrolysis as equal to the initial substratc con~ntration, Hydrolysis rat~ were atsetted by the appearan~ of the product=/anit time, Kinetic constants were calculated us!n= the Michaeli~.-Mcnttn equation and the l i n e r tnmsl'orm methods of Lincwcaver-Burk, The valaes for/,'.~ were calculated by aSSulnin~ a catalytic subunit of ~lg kDa, Three ~parate kinetic studies were performed usinl~ different enzyme preparation=,

3. RESULTS A N D

I

10

I

I

I

20 30 40 Retention Time (man)

I

50

Fill. l, HPLC analysis of peptid¢ products formed by incubation of endothelin 1 with rat kiduey membranc¢ Endothelin I (501vM) was incubatod with $ #11 kidney membranes in 20 ntM Tris-HCl, pH 8,0. for 16 h at 37"C in the ab~nc¢ and presenceof 100pM phosphoramidon (A and B. re'=peeti:,e!y),Then, each ~mple ~wasfractionated by HPLC as d~cril:~..d in Materials and Methodi. The product peaks am aumberod in order of yieitl, and numberl correspond to thole in Table ! and Fill. 2. Arrows indicate the ret©ntion tim~ of intact endothdin I.

DISCUSSION

Fig. IA shows tl~eH P L C pattern ofendothelin I and itsfragments after 15 h incubation with rat kidney membranes. Endothelin I was hydrolyzed by the membranes to four major degradation products (designated peaks I.-4) which w=re d~tected by fl~eirultraviolet absorbance at 215 nm. In the presence of 100 ,uM phosphoramidon, peaks 2--4 disappeared on tl~eH P L C prod!e, whereas peak l was una~¢cted and clearly elutea as peak l* at the corresponding retention tim= (Fig. IB). The individual peak fractions shown in Fig. IA and B were collected and theiramino acid sequences were determined in an automated gas-phase sequencer (Table I).The sitesof hydrolysis predicted from these analyses arc shown in Fig. 2. The resultsshowed tl~atpeak l and p~ak i',bo~h ins~nsRiveto pho~phorafnidon, wcr= idcn. titaland consisted of a mixture of Asp-lle.lle-Trp and the N-terminal fragment oftndothelin l, indicatingthat these fragments were generated by ¢leavag~ at the 304

I

0

Lcut~-Asp t' bond. Peaks 2--4, all sensitive to phosphoram!don, were the N.terminal fragment of endothelin I (peak 2) or the products by cleavage at tl~¢ SerS-Lcu a bond (peaks 3 and 4). A metallo.endop=ptidase recently isolated from rat kidney membranes by us [l3] is insensitiveto phosphoramidon and cloves endothelin l in a limited fashion at the Leut~-Asp tm bond [14].In contrast, another metallo-endopeptidas¢ from rat kidney membranes, ¢ndopeptidase 24,1 I, is known to be sensitiveto phosphoramidon [1,2] and to clctv¢ the hormone at the Sers= Leu ~ and Aspi"-lle)~ bonds [ll]. Therefore, the results presented above strongly suggest that peaks I and I' were generated by our newly isolatedmttallo-endopcptidasc, whereas l~caks 2--4 ~ pi=o~uo=-~ b-y ~dup,=~ildase 24,11. Endothelin I is supposud to be an important physiological substrate for rat endop=ptidase 24,11 since the

Volume 309. number 3

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Fill. 2. Identities of the products formed by incubation of endothelin l with rat kidney membrane. The numb=red pcptidcs corresponding to the HPLC peaks in Fig. 1 were identified by determininll amino acid =qucn=s in an automated ga,=.phas¢s=quen=r. Arrows indic=to the bonds attacked by the en~mc.

K,, value for ¢ndothelin I is amongst the lowest re. ported for this enzyme (Kin=R•3 ,aM, A'=,~=2.19 s", k~.,l /:.,=9.52 x l0 s s't.M "~) [l 1]. To assess the potency of our isolated enzyme in endothelin l hydrolysis, we de. retrained kinetic parameters for endothelin I hydrolysis by our enzyme and compar~ the obtained values with those by cndopcptidase 24,1 I. As shown in Fig. 3, kinetic constants were calculated using the MichaelisMenten equation and the linear transform methods of Lineweaver-Burk. Graphic analysis yielded values o f g , = T l . 5 /.~M, k,a,=l.4~ s" (V,=~=3.23 nM.s"; [enzyme]=2.17 nM at the subunit molecular mass of 88 kDa). giving a ratio of k,-,,/Km=2,08 x 104 s't'M'=• This K,, value by our enzyme for ¢ndothelin I is about 30fold higher and the [:,=,IK,,, is about 50-fold lower than those for endopeptidase 24,11, respectively. Thus. taken together with the finaings that thr~ of the four peaks shown in Fig, I A were the products by cndopeptidase 24.11. this enzyme seems to metaboliz~ endothelin 1 more efficiently than does our isolated enzyme from rat kidney membranes. In conclusion, this study indicates that endopeptidase Table I Amino acid f,:quen==s of d~gradation products of endothelinl Pc.al~ I 2 3 4 1'

Amino acid sequence

Cleavage site

Asp.ll¢-lle-Trp Cys-Ser-Cys.Ser-Ser-Lea.Met-Asp.Lys.Glu Cys.Ser-Cys.Ser.Scr.Lcu.Met.Asp.Lys.Glu. Leu.Mat-Asp-Lys-Ola-Cys.Val.Tyr.Ph¢l..~u-M¢t-Asp-Lys.Gla.Cy,=.VabTyr-PheAsp-tle-lle-Trp Cys-~r..Cys-~r-S~r-t~,J-M~_t-Asp Lyp~_I_u.

Leu'~-Asp'm N.t¢rminal Nqcrminal ScP-i..cu" SerS-L,ca * Lean-Asp 't N.~rminal

Th= individual p,=ak fractions, as designated in Fig, 1, were collected and their amino acid scqueac~ determined in an automated gas-phase protein sequen~r as described in Materials and Methods,

o

/

1/5 I

to

I

20

I

3o

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t0 so S(gM)

I

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(raM") I

70

!

00

Fig. 3. Dependence of velocity of hydrolysis by a recently isolated metallo.endol~ptidas¢ on cndothelin i concentration. Velocities for hydrolysis of various cong=ntrations of ¢ndothdin I by a recently i~lated rat mctallo-cndopcptida,~¢ were determined ~y quantitative HPLC analysis as described in Mat¢,'ials and Methods, A Line. weaver-Burk double reciprocal plot of the same data is shown in the in.t,

24,11 and our isolated rat metallo-endopeptidas¢, both as integrated membrane proteins, participate in the metabolism of endothelin I by rat kidney membranes. Thus. both enzymes may be physiologically important, and further studies are needed to elucidate their roles in the renal metabolism of endothelin 1 in vivo. Ar/,'omwledjteme~Jts: This study was supported in part by a grant from the Smoking Research Foundation in Japan•

REFERENCES [I] Kcnny, A,J. (1986) Trends Biochcm. ScL II.40-42, [2] Erdos, E,G. and Skidgcl, R.A• (|989) FASEB J, 3. 145-151. [3] Olin,=, G.M.. Spear. K,L,, Siegel. N.R., Reiahard, E.J. and Zurchcr-N¢¢ly. H.A. (1987) Eur, J• Biochcm. 170. 431..434, [4] Stcpb=nson,S.L. and Kenny. A.J. (19B7) Biochcm, J. 243. 1831~7, [5] Sonnenb=rB. J.L,. Sakan=, Y,, JcnB. A.Y.. Ko¢hn. J,A.. Ans¢ll, J.A,, W¢nnojl¢, L,P. and Ghai• R,D. (1988) Peptid~ 9. !73-180, [6] Syb=rtz, E,J., Chiu, P,J,S.• Vemulapalli, S., Pitts, B.. Foster, C.J.• Watkins• R.W,, BarnEtt, A, and Hanslanscr, M•F. (19~9)J, Fhar. macol. Exp. Ther. 250. 624-6} I, [7] Olins, G.M,. Kri¢tcr, P,A,, Trapani, AJ., Spear. K.L. and gory. P,R, (1989) Mol, Cell. Endocrinol. 61,210-218. [8] Danielewicz, J,C.• Barclay, P.L., Bamish, I.T.. Brown• D., Campbell, S,F.• James. K,, Samuels, G.M.R., Tenets. N,K• an~t Wytb=s• M.J. (1989) Biochcm, Biophys. R¢~.Commun, 164, Sg6S. [9] Yanagi~wa, M.. Kurihara, H.. Kimura. 5.. Tomob=. Y., Kobayashi. M., Missal, Y., Yazaki. Y., Goto. K. and Masaki. T. (1988) Natur~ 332. 411-415. [lO] Scieli, A.G,. Vija~,ara_ahavan, J,. Hersh, L, and C.arrctcro, O. (1989) Hypgnensioa t4, 353. [1 I] Vijayaraghavan• J., S¢i¢1i, G,, C.arretero, O.A.. Slaughter, C.• Moomaw. C, and Hersh. L,B. (1990) J. Biol. Chore, 265. 14150141S~i,

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[12} $OkOIOvlky.M.. G,iron. R.. Klooj. Y.. Bdohih. A.. :r~ii. F.E.. Blumbl:rl. S. ;lad Flcmin~r. G. (|990) Pr~:. N~th At, d. Sci. USA B7. 4"/02-4706. [I :1| Yam.¢uchi. T.. Kido. H.. Fukasc. M.. Fuji|.. 1". ~nd Katunuma. N. (1991) Eur, J. Biochcm. 2QO,563-$71,

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[14) Yam.Buchi, T., Kido, H. and Ka|unun'm, N, (1992) Eur, J. Die. chem. 204. M?-S$2. [i5] Siephcnr~n. S.L.. and K~ny. A.J. (|98?) ~iO-¢h~t:mrJ. 241. 23%