Rat plasma kallikrein: purification, NH2-terminal sequencing and development of a specific radioimmunoassay

l~i~hi~xa et BJ~Jltrsic~Acsa, ~r'P~l~.19B9) I~)~- Ln) 121.~'+'imr

]03

Rat plasma kallikrein: purification, NH a-terminal sequencing and development of a specific radioimmunoassay Joanna Pa~uin s, Suzanne Benjanne] ~, Nicole Sawyer t,. Claude I.azure :, Mid~l Chr~]ien ~ and Nabi[ G. Seidah .flA. a~~$ ~ Zab~q1~Ns ~q- +$i~-hemica¢ ~.nd ~.4~,st,ee~a," Neu,r~d~l~nOtogy, Cl~,rica~RLl~e~m#~tn~t~t!eof ,~onz,~'al. Mon~at ( Cana~j

IKcc~'P,'~d17 July lb'~,,~}

Key~.rd,; K~lli]~n; An'~no ~;fllirmL ~qo¢0ce: C]cav.~¢ p~ttetera.~ Radioimmano~a~':

Affimity ]~e]~A tracer: fRaz pl3~,ll;l'j

R.~t ~h~ma kalllkrdn (rPIf0 wa~ pro.re'led 4o homogelleil~ |,,ran pla.r.m~ t~iBl~ ~ffhliey and hieh.l~dormam-~ Ik~id d h r o ~ o ~ q r a [ ~ lechmlque~ and mbjected to l'qH:.lemthud ~qt~n¢img. The data shor,~d shat the sequem"~d ~eg~erlts ~[ the ~ d ~ l o r y ~ j ) ~ c,~l~.~ti¢ (]iRh0 ¢haim of the p ~ i m t ~ , f e s t i v a l S , displa~ 73 nmt 9Z~ ~qu~en¢~ ~n~laril~ ~ ~ ¢ o ~ t e r l ~ t in lmma~ ~ Itall~-'eim, ~ sequela bmnoto~, in c~mjunelinn with the d~mth~l nml~" ~ and inh~ito¢ ~nsilhq[y ~ o r l II~ idl~n¢il~/PIPI ~ isad~tt~l ~t~yme as pla_sma kalllkrein, l'ad~oimmmloa~'~y was deveinl~[L Since Tyr-iodinal~l ~PK ~ nml t~reg~iz~l by the anfiaemum+ two dsemat~e J I p l ~ h e ~ w e ~ Iomld Io I ~ meoe~ul. These i ~ l l l ~ ~ use of n 13~er ~ t h ~ of rP~ modified ~fth ¢icher affintt~ eeagem +~l-I~,led DT~pr-Gin-Ph~L~s-Arg ddorome: d ketene or with the Belton ltumer reagent. Tl~e u~aJble tinge of llhm ~ is b ~ ' ~ n 15-I~1~ |m~l per ~1~. The mntl~m~ ~a~ sho~n t~ Idad ~ m~memleri¢~md dlme/ie of tlPK. D e ~ oil Ilhe enm,alne in s~Jium il~tlec~l stdf~ke ~ not ~holi~b immu~e recognillon a~ly ~s IOllR as the regalntory sllbunit b ntlaehed I:o the e~walylle chain- (P~.idadmnm" fe~lSt~]¢m of rPK n~ulL~ In eorn[~lete I~s nl immmMmm~idl)r. This ol~+¢nafion mggests that peH',aps ~ dls~dflde I ] n k ~ 0~ the cad~l)'fl¢ and regdsmq. P o l Y p e I ~ ~mlehew b~]t~ to ~ the nnlligenic t,~itope fre~ denalxn',a~n, Ahen~tlvely, the eFW~L~pe(s)recol~lted by t l ~ ~ ' b o ~ ~ a domain whidh ind..~le~ both T:pr and Cy~ I~si~lle~ ~ for in,mr,me Rco~th~en, InIPoduK~k~ Pla+mt~kaiHkR+n is a serimc pro{cinaxe eh~eulatmg irt the pl~rna ~ it.s +ymog,eP c+lted prekallikrcin. The zymogen i~ activated t]~rough limited pxOtcolysis primarily by activnted facto+" XI[ [1] ut posslbty by itself I2]. The ~ r v e end5 me displays a trypsin-llke activity and i~ known to proc¢~ ~verzl protein precurSOrs by limited Abbt'~Olrd: KMWK. Idllh mok~lar w~ghe tdmm~gen; rPK. tat plasma k.sllLkmein: HPLC. K[gh-pre~,are liq,lJd ¢h/~J~trJ~Fal~hc.". SD~, ~tlium ~ade~'l ~ullal¢, PAGF~ i~lya~,'rylamide ~ ~l~':tr0phore~]~; TLCK. N~p-tO~yJ-LI-ys ¢hloc0~tcJh~l k~le~e: YF.FKR-CK. i)TjrLO]u.L]Phbr, Ly~-u~.~ chlnr,amtlhyl ke¢one; L,~'J'A, L"t[q~rl{IT~L,~l~iEl¢~ -

~lm~u!fO¢.lc mcid; ~ 2-[bL~'-h~trc~x~f¢lhyl).ammo]eihan~m~ic ~ i d ; ]~zSO, din~lhyl ~a~lfo~,id¢: Z, b~rff_yl0~)¢~rbo.yL: A/d.C. 7aminoJ~nneqh~fk~tLmadn; clDNA. ¢4~11~¢11~1~L~1~-"~NA; RIA, rm]k~

WO~c~ly~is. F~ examF,le, lhi~ enzyme ~L~erme~ the v~soactJve peptidc bradyki~in Cram hig]k molecular weight lonhaogen (HMWK). acli,'al~ |act0¢ XH during the early phase of the surra¢c-.depcndeot intrinsic palhway of b t ~ 1 0oagulallolt, nnd panicipate.~ in the activation of plasmlnogen to plasmin dutS~: fibrinol.~i~ I1|. H M W K helps in there .~ti~ nf reactiom; (1) by acting as a co[actor vchJch increase.~ the ratem of lh~u reactions and (2~ by anehoriag l~rckallikrein at ~ite~ of reaetlem thr~yJgh its ,~w~rl interaction with ec'~atively charged mrfa~-s [l]. Plasma kalliktein ha~ at~o been psepc~sed io part[cipnce in the tx}nver~ion o[ prm'¢[li~ into r~nin in the ~nin+aagiol©nsm s~tet~ !.~.4] al~d, in addition, to participate in ~c ptot~l).tiu pro~esslng or p~ohorrnone~ on the ba.,~is of tl~ ¢le'~vage pattern observed in viire using propeptide ~.ubslr'alm, ~ f5.-91.

imm~a~may. +" h ~ur la~ratory, plasma kallikrtin L~titi0d r , ~ et~lxer~0r¢in¢ or CturT~zl:m~ P.~; M. CP,r+I~n. J.A. de g ~ i.ab,ot~r~.,i~, ot Mohx~l]r Ne~o~mdo~d~01ogy. Clinical R~s~ardl I~litm¢ oF MOndreml. lift

IT'int:Avcnlm ~ Moncr~d, Quebec. Cmnad~ H~¢lhI 1~?,

E.tII~ [I~,CM-~cein~ p l ' ~ l ~ " 1 a"til Mn~tuml and i~unn~T~O~J~l uf.als'~t.:~r+w~t~d lh~' ide+14ir-aLit,r.lmwetin"~ l h ¢Iplt'.yIiI~

In order t~ establish the TOlerance of tllese additional ro]es f~r plasma kaliikrein in vivo. it i:~ necessary' to study the physiological regulation and ;~alization of the enzyme. Therefore, we have produced an a~ti.~zrom directed against rPK in order to devoMp a specific assay for delo;ting tl~ ¢neyme. The mt s~lem is of partiealar imercst, sin~ it has served as a model in many hypertension and pmholmone biosynthesis studies. [n this work. we report the purification of rPK. The identity of the enzyme is established o a the basis of its molecular stmctuce, ermymal~ propertit~ NHz-temll. hal sequence and inhibnion profile, ill addition, a highly specific radiolmmuneassay was de-,'¢lopped using as a tracer the en~'mo modified with the affinity reagent =~SI-YEFKR-CK, or by the Bollou Hentet proeadur¢. Material ami Medmds Purificaoon and enzymatic unsay of rat plasma kalltkrein Blood was collated from tats an~sthesiz~ with l~ntobashita] in the presemx of 1.55 (w/v) EDTA ttipotasmum salt (AId=ieh), Plasma was obtained from blood by otmtrifugation at 1400 ~¢g for 20 rain and st~ed at - 30"C. rPK was purified from froaen plasma essentially as ~ H b e d for the isolatlo~, of fire porcine plasma kallikroiu [5], with the ex~eptJ~'~ that elution from the STl-Agarose column (10 nd gel) was performed in ttm p~e~¢nce of 5 mM NaOH and. 0,15 Brij as dog'ribed by lqagase and Barrett [101 and not in the p,mseace of ] M beatZamKline, The daate was ¢ol~ected a~ 0.g ml fractions into 0.2 m] of 500 mM Mas, 5 mM EDTA IpH 6,0) (buffer AI- The two active enzym© pools obtained were dil~tiy Co0nceatrated in a Medal 320 ProDiCort r~'fiea'txamcemtrator (Pierce), dlaiysed aT~ins~ l i d vol. O[ the storage solution (10% {v/v) b~ffer A, ~O $ (~/~) 8 i y ~ L ) and stor=d at -20'~C until further ana]y~s. Prior to nfieror,e~oe'ncin~, a p0~'= tlon of the eor~c..¢atta=.¢d pool 2 (see ]~esulL~) was dia ] y ~ against ]3 p.M SDS and cono~ntrated approx. 100-fold in vacuo. In few u~s~s, the STl-Agatos¢ pools wcr~ further analysed by HPLC siz¢-exeluslon chromatography using a 7,5 × 600 mm Bin-S11 TSK 250 oninnm (Bio-Rad), Elation was perlormed in the presence of 100 mM Mes, 1 mM EI)TA (pH 6.0) at a flow rate of 1 ml/min, T l ~ pooled enzymaficafly active fractions wca:e rhea dialysed and oonveatrated against 50 mM NH4OAc~ 1 mM EDTA. (pH 4.75) and subjeered to micxosequenoc analysis. Enzymatic uctivity was auma~-ed by monitoring the fluorescence of ~'eleased.A M C from Z.LAla-LLys-LAIgA M C [11 I. The assay mix contained 100 mM Bes ( p H 8-5) 1 mM EDTA, 5% (v/v) M~SO, 42 p M of the flaoro~nic substrate and the enzyme, in a total volume of 1 ml. One unit of enzyme activity (U) r e ~ 1 nmoi A M C per rain m room temperature under these co~li-

tions. "The effect ol" various inhibtiors on enzyme activil'~ was deluTmined as n:ported previously [I l]. Sodium d~Meo,l sulfate.polyacrylamMe gel elecmopheresi~ 10~ poiyacrylamid¢ slab gel eJoctrophoresis in the presence of S O S was performc, d essentially as per Lacmmli [12]. In order to assess the positiono1 r P K folg0wing SDS=PAG~ the enzyme was prior labeled at its acti~ site with ]~SI-YEFKR-CK [6.111. The conditions [or the clectxophorcti¢ separation and the pm, tocols used to ~ 1 the protein binds by au~adiography have been described elsewhere [7.]1], Mieresequence determination SH.~-terminal amino acid ..sc,.luenCe analyses were pcrrormacl using an Applied Biasystems gas-phase sequenator [5,]3]. Phenylthlohydantoins obtained from each cycle ~ analysed by an on-line model 120A RP-HPLC as already described [13.14]. Preparation of anfi-rPK antibody and development of u ~oeclfJc RIA Antibody was raised in male New Zealand {2 kg) rabbits- For each injection a total of 1 pg of pudfiixi rPK (pOOl 2 from chromatography oven" STI-Agarose) was emulsified with complete Fmund's adjuvant (DIFCO laboratories, Michigan) and was iajectod both sul~atineously and intramus0ularly, A total of five injections were made at 15 days intervals, The tiler of the antibody used was 1/16000 fitml dilution, For RIA we used purified rPK (pool 2) as the antigen and the amti-rPK antibody developed as described in the abo~e seetkm. The tracker w ~ elthet rPK reacled with the l~[-lab6ed peatapeptide ehlmomethylketone [1l] and dcsalted on a column of 0 - 5 0 Sephadex (100 x I cm), or rPK couylcd to the =ZSl. labeled Bolton Hunter reagent (DuponO as specified by the manufacturer's instruction,s. Samp]~ (purified rPK, pl~Lsma or serum frum a number of species, etc,) and standard were irm'ubated with the tracer (6000 upm) and the antibody {[-L11~Idilution 1/16000) for 24 h at 4 ° C in a final volume of 400 ~1. containing S0 mM Na;~HPOa (pH 7,4). 10 mM Na2EDTA. 0,2% Triton X-100 a n d 0.55 human serum albumin, This was followed by the addition of ,500 pl of immol~liz~ anti rabbit IgO (Bioproducts) and a further iaoubation of 30 rain at room tempmatum. After the addition of 2 ml of 20 mM N % HPO4 (pH 7.4), the iucubation mixture was orutrlraged at 2300 × g for 30 rain at 4t'C, and the re~.u]ting ~'~let was colnted on ~Ln L K B gamma counter, model 1274. Analysis of the slandard o.trve using this R I A indiua,tus an EDso value of 60 fmol (5.5 rig), with a reliable detection xanSe of 15-150 fmol (1.4-14 ng). Plasma kallikrcin R I A was ais,~ pudormed on samples sepasated by SDS-PAGF., uame.,ly, rm plasma (2 ~l) and rPK pool 2 (30 p¢'..ol). After e]ec~rophor¢~s, individual

105 ] a n ~ o f the gel were frozen on sollti CO 2, cut into 1 m m slic~, and cluted overflight at foam ~mperatr, zre in 250 gi RIA ht~ffer (see above). R[A was then pcrformcd in duplicase using 90 pl aliquots of the e]ual.es. As controls. :ed,c~xt and ~on-rednc~l rpK pool 2 ]abet~ with the ~I-lab¢l~d pemapeptid¢ thloromcthylkttonc (400{]0 cpm) were also run in parallel, In this case. the radioactivity ~nmtained within the sllces at the control lan~ were counted directly ir~ a gamma counter.

TABLEIt G~wm~(.,,~ <>]fA,I o¢.[¢=,Jf~.ltttdfRJea~ihi!~Tpr=.J¢l=,~njrq&;om'in('aed ltw~anpl~z, makaff~&re, et

(A)s u ~ m e • ~PIle-Phe-Atg-.AMC Z-A[a-I.ys./~rg-AMC o~:r. Pro- PI'N-.~Lr~.A MC Z-Ata-A,~-Ar~-AMC Z-ALa-Ly~-Ly~-AMC

a~ v 3.26 1.r.~ tL03 0.1I 0.05

p~ " n.4J.e ].L~J U,?~ [J.42 022

Z-A[a-Lou-Ly~-AMC

(}.05

n.d.

P.esalls (B) Inhibitor

Pvri~ea,i~ ol r P K Tnc b'l"l-Agarose + reverse-pha.~e HPLC protocol deveioped for the purification of porcinc plasma ka]likr~m [5] could r~ot directly be applied to the rat enzyme due to a ,~ry low r ~ o ' , ~ of protein in this particular ca~e~ The NaOH + detergent solution w3s found to I ~ aJ~ efficient elcant and, in contra*t to benzamidinc [5]. allowed the monitoring of enzyme activity in the eoluran effluent, A summary of the pmification from rat plasma incorporating the modified ¢lutioo p~ocedure is shown in Table ], TWO activity

p e a k s e l u t e d from STI-Agarosc in tbc

preseno~ of N o a H : an early peak (1) and a late Peek (2} (nOt shown), The pool Of active fractions under e a c h peak displayed similar activity ratios with I]uorogenic AMC-suhsttates (Table IIA} and a similar inhibition prefil¢ (Table liB) to ti~sc tx~ported for por~ne and human plasma kaihkmin [~], The~c ~sults suggest that piasma ka]likrmn coot.,ibutes majol~tatliy t o the activity o f these

pools,

TABLE I

Fr BCliO[X

~°~lein ~ AelJ,~ay ~ S~,ecJ~ie Acti~1 ty (m~) (areal/miTt) ar~Li¥iL3 ~ yield) [areaL/non

p~ m# Idilt,~engfitu~allna SU~mal~i

S t4S

~9

0.D]

Phcnyl 6ormla~e I M fJ~a~di#c

o

218

~.4~

I[

l~

?5

2 fi48

3~

1@9

0.62

97,5

1 ~'/4

,~'

&14

72.2

I $oo

to

mtmSephe~0~ N a ~ gr~ien~ $13-~ 5 mM N o a h • poor 2

pool 2

• Plamtaka~tikrein w~ obtained [r~mr, ]4~} S ~ ' =-atLal~rt~, a ~

~ll~umin ~ Ih~

r~Lac~dard,

"l~c ~latic ~etivit)' and protein eo[~te0( *¢r© d¢iermir)¢~J ~n the d~ly~oS[¢oet~on. • The tn~am&t~z ae'Li,,'Lcyand p~,l~n COnl~0t ~¢t~ ~¢ltnmiftcd aCter Ih¢ fi~ e~tr~lion/dial~ procedure.

Final c~necntration

• iohibitlon" tal h humaJt =

pig =

iX] n~m ~,~ SS =it I.O0tim 2~ 29 68 0.2m,M [~ e t) 3.2mM 0 4 0 ~.(11 r0M ~6 S2 SS Leul°¢Ptin a.t~lr0M 94 9'7 81 Pep~t~tln 0.01r0m 0 3 0 STt 0.5an/mr 9c~ ~,~ 33 LDT( 100 r,~/mt 0 a o YEFKR~Kr 1 t,M 0i ~z ~9 ' ]oJtm[;at~ ~rt¢ mea~u~,t a~d¢~ibed, u~,l¢rM~LeH~I~and MecJ~DFP PMSF 1e¢oao:-Litacia TLCK ,~l~[iparq

~

u~in~ 41 te ~2 pM m" the iMi~al*d ~uL'~ualeaM ,-~rngarea t~

the initLMca'."~*~trvedto Ih~ p re~wlO=uf Z-A]a-Ia,.s-AtS-AMC. ~ Similarrc~,~lt*,,,~ ,:,bt~uc,*~ ~sir~8 tire~wc,STI.Ag,a*ompaean. • Thepm~Pea~

human ¢°2Sm~'~'¢r* ~fifie~]an already de~cHb°d

151. , The ¢nz3qme W~L~prri~¢L~bale~ with I1~J~hibilor t~r 30 mi. in tO0 tuFA B~. t ~ M EDTA ( p H $.0) l~t'o~e the ~ddition at the ~LL]~strait. Voll~'L~g the 101~llttubatM0. pt'fit~l tlt~ ,~.(i'vil,)" remainlrlg I~laliv¢ to the om~tro[ t~yn.~ I:~"~lar~ul~ta~l without iaSeilor t was mem~ut~r] USlng Z-Ala-L)~-Arg-A'~[C ~Mwa~* in tale ahq33-~.~15 described Lmder Ma~caials and Methods, r The inhtl~ito~ wa~ pretnoabltl*d with tb.e ~J~yme: ,~,~the eoneenttst•n indicated and Ihen d t l u l ~ 12,5-f~]d i~t ~.1~ a$~ily {~,utfer.

The sql-Agaro~e enzyme pools were analysed by SDS-PAGE following their affiniLs' labedng with L2StYEFKR-CK. AS atr~ady reported with the porcine enzyme t.~,11], thi~ rcagcot blot% to the a~tive Mte of tb¢ protein and permits to visuahse different [arms o f the cmalytic ~.haln on autoradiograms: a reduo:d ~:hain of 34-38 kDa and a chain disulfide liP.ked to a companion (regulatory) eh3in(s) forming a cocnpl~( of 88-89 kDa [tl I. Labeling was performed under conditions which inhibited 100~o of the activity memu~ed with Z-I.Alat.Lys-LArg-AMC [l II, The subonit structure of the e3~ectrophotesed enzyme from pool 2 clearly t~cmbles that ofporcine plasma. katligfein, with the exception that tl~ radiolabelcd spExi~ misrateO with ~ slightly hlghel appazcot molecular ma.~ than their porcine counterpart, Indeed, the apparent ma~ o[ the rat complex and catalytic chain a~, respectively. 91,3 kDa (Fig. 1, - S H ) a n d 41.3 hDa [Fig. t, + SH), Micz'oseqoencing analysis of pool 2

revealed an hllmogene~ms enzyme psep~r,~fion ~s~ heiowL indicates the psesenc¢ of more than one complex [51. 60. 62 and 91.3 kDa: Fig. I, - S H ) . Howe~,er, all ~;omplexas an~ formed by a disulfide linkage involvlng edhcr a 41,3 Or a 37.2 i D a c.atal),tic chain (Pig. 1, SH), The exlstcncc of radio]aheled bands of similar apl~l~Wil molecular mass in both i~ols, would suggest that the addifianal hands present in ;xmi i are due to de~aded forms of the enzyme, This hypothoois is sup= po.:e~ by the similas activity and inhibition profiles observed with both pools, [t is worth noting that the relative importance of the additional tadiolaheled bands of pool 1 varied Irom c,i]e pievarafion to another, The STl-Agasos¢-activ¢

pools obtained

ft~om d i f f e r -

prclmmtioPs were further analysed by HPLC size exclusion chromatography, In most cz~s, the enz2orne behaves as an homodimef elutiog with an apparent moic.~t,;aJ ,,lass Of a~:~ut 180 kDa fFig. 9). However, microhetesogenclty was observed at both the protcin level {by mca~udng the absorbance at 230 rim) and the enzyme level fhy measming the activity) (Fig 2~. This cot

l

2

1

2

lD

S~ D

~ ~

25o ~ 2oo ~ ~ :~ too -t ~ ~ ~o

~_~==

~ ,~

/~ I

\I. " ~

c~ ,.~

r-

~" ": .~

toF'mctl~n Num4~lr 21;. IO-5 en,)3o Fla. 2. HPLC.f~!. fdu'~tian or rPg. C~el littranon ~ the ST~-Aga~ose fsoo] 2 w&~ I ' ~ FO~nCdcm $. gio-Rnd B i o ~ i l TSK-250 c o ] a m IfaD0× 7.S mrs). Abml.~0uto¢of the ¢olumt~ef~uenl w'&sematinlm~r,~ monitored =~ 230 ¢~m ( . ). Enzyme aea~t~' ( I ) W~LSm ~ . ~ r e d [or each f ~ , o n ~il~g 10 psi ~liquoL e l , Lh¢ I.~ds e l acl/~ity imotil¢, rF'K

¢lulcs~s a dimer*dh a*t appatert[ ¢no]o~,JlarIr~s of I ~ ED~II~ ~l~gn'°x'~SDltshcqdtl°" ~ikelyecprcSCnL'Lhemgmcmetief°rm°fth¢

enz~,.me,Tl~arnt~ciamole,:~at massma,flltec~arc: *.hyroglobalin(670 kDa), ]mmun~lobulin O t1~,8 kDa), ovaJbumin(4~. kDal and m~glol:./, If? kOa), microbetcrogene~ty was more v i s i ~ u~h~g the easlierthan t]zc later.elating pools from the ST]-Agarose culuran. This additional HPLC purification step was routinely included in ~der to isoSate a pure ectzyme from 13oo] I[ foe Lhe productlo;1 of an antibody.

M/cro,vequeaee a,ai)'sls

--

¢-H

+SH

FiK. I. S I ~ - P A G E of TPK. R¢~3xgh.,~y. OJ and 0.0~ U from pooLs 1

and 2 obt~J~l [ollowi,i; STI-AI~0~¢ ¢hromt~taphy *ve~ la~tl,~ wan I~SI-YEFKR-CKa n d stt'~e(~gl to ¢l~tf01~hol~i6 ir~ 111¢ab~rK:¢ (-St[}o[ p~z~nce(+SUtof 2-~ptoeUtanol, l"~¢ resglting a,toradbog[ams are ~ . L o e ~ r ~xp~ore 6nI~ did not gveal ".AlppkII~nllt~y" bands. [qumbeys ] air0. 2 ~ 10f. Or S~tl¢lar~s ¢ott4::s~od to the id¢naly rum'o.er of t~t¢ alaedysot pool Ph|/tlh~ul n~o]t.~dat m i ~ *~rda4~l~ ( $ T D f in i d a arc slU)Wl'* i¢1 tl~ arsenal ,.ides of the

autor~o~L, ns-

NHz-terminal sequencing of the STI-Agasose pool 2 from rat plasma fT0~ of this pool was mtatysed) se*,~ealed the preseno¢ of three polypepfides v~ich oould Ix: distingaishcd by their iodividual initial yield (rasf,~ tiveJy. 3~), 26S attd 132 pmol), One chain (390 pmol) begins with [le-VaI-G]y-Gly and shows an NHz-termin.al sequence similarity to the fight ch#n of porcine a n d human plasma kaliikrcin (Fig, 3). It must bc noted that in some batches of the r P K a low yield Asr/' was observed upon mierosequet~mz, suggesting partial #ycosylmion of this residue which is located at a patenflu1 site for such a modification [16]. Glycosyiat~d Asu wouM not he eattacted in the sequenator solvant [141. The deduced s~qncJl¢~ of the t',~. semaining chains of the rat. enzyme were. restively, Gly-X-l.,eu-Ser-GIn= L~-Tyr-Ala-Asn-Thr-Ph~Phe.Arg-Gl.y-Gly.Asp-LcuAla.AIa.IM 0 3 2 pmol) a n d Gly-Gly-X-L.¢a-Ala= Aht-[~,.Tyr.Thr-Pm..Asp-Ala-Glo (2fi5 pmol), The two ~'laing shaR ~ identical seven-re,idea segment (as ondcr]i~ed), suga~.tlng they belong to a omrm'ion polypeptide. By c.ccnbining sequenc0 data from these, chains. it was found thai the sesulting polyptptid¢ shows ,s¢q o e n c ~ similarity to the heav~ chain of human and porcine pla_~ma kallikr~n [Fig, 3). ]dentlca] results have I ~ 0 n o b t a i n ~ l b y sr,q u c n c i n g c/.thm- p o o l l p r o s e i n a f t e r

H P L C size ¢~¢lusion c]lromatography or pool 2 protein

107 from three separate rat plasma preparation, Me:reov~. the: rat protein partial sequence was independently confirmed by seq~ncing its corresponding eDNA [17], The mieros.equencing ]0esuIts revea]ed tl~e ~rl~nce of only one sequencab~'e protein species, suggesting that the erlzyme preparations *,,:ere virtually homogeneous. Based o n an estimated molecular mass of 91.3 kDa (Fig. f ) and the amount of material subjected to mjcrosequenclng (70~ of pout 2, Table I = 98 p g = 1 nma]), the sequencing yicld ,d" 400 pmol translat~ into a 40% initial yield. An NH:-termi,~l cleaved form of plasma kalffkrein regulate:~ chain was det~ted in relatively ~arge:amounts in our rat prc'parations (265 pme:l or 66%

4C~ R(F ~ ~[.Ll~.P[ikJ ~

200

O,cr

o,1

to

ro

~o

AI Ftg 4, PL~e,l'~'~ k~.llik~'eie ri,~ioimm,pn~ai~ay. I.ines I~:prt~l dixp]ac~rr~nt ¢ o r ~ usil%g ~l~.ma ebta~ned r¢om htsma0 Iol, rat to), pLgI'll and ~.mm ~-~hLai~_,~dfrom hoarse {~.) ~ d [elm cuff (o). The dSsplat~',Iffl~r~t ~Ul'3~ USi~B ~11~]'i~2~ ePK. is i~eluded (Pout IlL ~ ab~Js~

e~l~3.e_~ of ?)a~ni Xa]llkreln a~,~¢ sirrahs

aeplet~ the i~l~-~a or ~rum volame (in ,~11 takctt f~- di~pl~'mcnt ex~ays or [he v~cdumrof puritiM q:~K~ ' S T I - A ~ ~ 2 c,m~taJmrtg ]~O I'at~l/ml oF ea~qttencab]e ~ry'me a~'t~- IIPLC" gel allratio0)

Izg~L~¢qt~- t/d~pa~'l chz~ln S

LC

15

Fc~ the slandard di.~pLaeeme'at rmr~e. The ordinate t~preser,ls ~.he mtml~er o[¢pm or tz~I-YEFKU,CK Lab*k~ rPK I ~ L,Othc anli h~y,

20

25

20

Itl

t5

o f ~[uencab]c regulatory cb~dn~), A similar ~le~-ved fe:rm wa~ al~o detected by micm~cquenuin8 a h~man plasma p~kaLlik|'~iu pceparalion (a gilt f r o m Dry, K, Fujiklawa and E.W. Da+ie) al~d represented approx. 30% o f seq~e~e-able regulatory chum (not shownt, I n ~ t h e.~es, these forms are produ~.x:,d,by a cleavage o¢~urrin:~ C.terrmn~l to the: s~.q,.tenee Pttetz-phe12-Argt3 present in the he.avy chain of p l ~ m a kallikrein.

~tl0.]~CZle ~LJfik'L~. eha~rl 5

~,PK lie vat GI, GIy Tbt ,~r.~hSefSet]~IGly Clu ,tp [~ro7z~ :la plPK ii~ ~'#i GI~ r ,1~

"Atr[A~i~ls~r!Ph~llAeu

17 I

'"

r;qu

Pro

l;In.

"~al C~O"G1y Thr /,~n S~r set k~u c)y G1%~'rp ~r~ It9 GLr~j ~==_. . . . . . . . . . . . . . . . ~__. = .. ~Q Z3 30

I "' "°

~]n AIzlI['yflN

''~°

I

~in K'a3 II''~"-t~'-~'Xz
Fig, 3. C~:4~p~n~n o( the NH:-~rmiaal m~irto ~'id seqeo~t~s o{ btm'~e (h.)~ bovine {b.), I~DrcLn¢ (p,) arid Cal (r,) plasma kaaikreia

(PK) for both theirregtdato~. ~ d ~Ualyii~ ch~tiDs.~hg sequt~t:t~ oF ~.PJl~b.PK a~l p,pK ~uem.~pectiv¢l~r~taken from L'hung ~ al,tl5 ], H~imark aod Damerll ned r~ia~ eLaL rs] Idenfiee] i~aidue~el~ plaoed .aJzhiabm'~swl'dlc any unid~lifiedaminoacidi~r~Macetiby Xxx, Byexpre~i~8 seqt~an3ehomo]o~fas ~h¢ ~ ~ idmlitmlresidue* the ~allalyscdrC~dUL'~, the P~BXllUlOOr ;beia o~ rPK ir~ ~espectk~ly, Sinularly,I1~e.:~lal~,i¢chin F the r~te:~zyn~i~, respectivety. 91and ~3~ homulol~.uu*,othe html.~ ~Ed~oqr~nee2n'~poeding el'rain. A dr.m~df~ll~ ~r ih¢ c~*lpl~lo/~ chain w~ r ~ l in hame~and ral ~ul aol in potciae enzymept%~pdo[s: a~inddeai~l~ the a,r~0w ( J,p16e NHr~¢twda~l ~0ea~e or IhL~clea'~nd [C~al b~]rls =ilh tl~ Cly tesidlae omqPeslmmdLngto poulk~n 14 in the u~¢leaved counterpart. The ~onbel t~r)iodicate~~ pot~a~tia] M-glye,~y]ationsite.

Rat plasma katlikrein RIA and SDS-P.4GE analysis As shown in Fig. 4, a RIA has been devele:ped with tPK antiserum usJng non-modified and ~z)I-YEFKRCK m,,xtified rPK us a standard an& a (racer, respectively, The RTA has an EDh~ of 60 fmol to rPK with a reliable: de:ruction range ot 15 to 150 fmol per tube. Rat plasma cxhibitad a parallel displacement of the I~ltracer bound to the zmti~rum, with rezpcct to the rPK standard. Mmue plasma re~lted in a non-pa~atld displa~.~'me~t curve, In contrast, pLatsma e:r serum f r o m eithex human, porcine, horse or fetal cult practically showed no cross-reactivity (Fig, 4), Ffxym the alive: data, the estimated ct,ncemtraLion 0[ immaunorcaetive plasma kal]iktein present in rat plasma is 0_6 nmol/ml (7.7 rag/140 m]). Although not sbowm the: R[A slandard displace.meal curve obtained under identical cortdidona but using rPK coupled to the ~:~I.labek-~ l~olton Hunter reagent, as the tracer was identical to ll'~t departed in Fig, 4, sug~e,~tin£ the ubserm~ of a critical Lys residue neoessary for immune r~cognition.

g

Spec/fict;y vj the"rPK anmerum The specificity of the rPK antibod2: ~'~ o~re~tly tested an ptasma. A combir~tion o f R I A and S I : ~ P A G E analyses showed that the antibody w~ognizes a single 91,6 kDa molecule in plasma tFig. 5A). The purified rPK pool 2 preparation also contains a simi lvrly sized immunoreactive ch,ain (Fig. 5B). Howe,,~r, a~ eddltiona,t immtmoreactive p
immunoreactivity, indicating that redueing or o~idizing agents cannot be used before electropboresL~ as a means of sclmratin $ the disu]fide.hoked calalyttc ~md regu. lator'y chei~s. However. the SDS-PAGE pfo['ik o b lained afler redoction of the ]z~I.YEFKR-CK-Iaheled rPK pool 2 (Ftg, 5C) suggest~ that most o f the catalytic chain is intact in this prepaJafiot~ and the desradation (Vq~ 5B) probably ,xo.trs within the t ~ u l a t ~ ehein. Therefore, the strateSyu.~o8 dcr-.atoring SDS-PAGE in combination with RIA could not he tu,ed in order to investigate whic~ of the two chains of rPK the antibody

Fi~ S.RIA.~D~-pAGE a m i ~ o[ rF~.I~I ~qm,~ (2 pL pand A} /~d ~!~i"l-Af.ltr4~.pool 2 (a ~ffet~et prquualkm f[om Zlm'~ur~:l in fig. L i:mm¢ls B.~ and ti) ~ ~bjected m SDS-PAGE i~ :he abum¢c (a~ B, D) ot in d e ptt~¢m¢ ~C) of 2-metr.aptocdlaJ~l. For and the rJkz~ c~euimxl 6ram lhe I ~ w~teB/ng the unnU:x~f~d mv0r:~ mmpk~ ~ ~ by RLak(A. I))~w)ik d~c nnlk~d~ity ~mine(1 in the slim~ of owym¢ rumples modirtcd with n=llY~aIISam eM~'q~l as i'a11~i~ ~ oi" immtm,0~t~i~e rFK (A, B} c¢ a~ kcFm/s]L~ of ~ 1 ~ 1 nu~'ial (C, D),

? " ~ 31

U 21

0

S

J~l

1~

~1

25

Elu'~ion Time (mini.) Fig. e. BPLC-~I fi]L~fio~ ~f tPK caL~yO~drain. G:I l"tILrallenw~s ~farmed ~ ia~i&m,.edin 111¢lef~n~ ~ Fi& ~. bm u~.~l~the t M

St~a-~dine,HCI pool of a ~dfN©mtpls~IN[t~l~J~~ B~Lll~g ~4"I~t~l~ (20(]0 U/h). Mt:az~rata0eSlSof ab~ano~ el 28Qnm ( , or-line
I'orm o4[" r ~ K , m i l e r [L i~ IlllIO~'l~l~.]¢

by the cMor
rad~cJabdedme,gentand misr~e~ .r~Ocrno~.t~luci~g condltk~s on SDS-PAGE~s a 91 k ~ i ~ - ' p ~ d e (not ~o~m). Ph~trmama markz~ pt~4~m m©; I h y z o ~ l i d (6"/0 kDa~ i m m u n ~ ] ~

( t~.IBkDI) and fil~nacle~(13 kDa).

recognizes, nor whether the antibody exclusively recognize~ ~.he dime~c form of p l ~ m a kalllkreln. Fortultousi% in one of our etlayme p~,paiatlOns obtained from a reJativdy oldex plasma collection we observed large amounts of mooometic rPK both in the 1 M 8uanldlne. HCI pool of the Phenyl Boronate column, or in the STl.affinity column p0~l 2 (see Table I). As Shown in Fig, 6, representing the H P L C gel filtration of the l M $mtoidine. H C | pool obtained from this preparation of rPK, the 180 k D a dlmcx and the ~ k l i a morn)mar detected by activity me.~so~ments were e~ually recognized by the r P K antibody. This conclu. sion isfurthex~ p p o ~ e d by ~ ¢ factthaton S D S - P A G E this goanidine - H C I poo~ was shown to exclaslvcly consist of the two chains form of rPK, This wen done either by RIA of the material ehitcd rTom gig slices obtained after SDS-PAGE of the whole $mmklloe. HCI pool, or by SDS-PAGE of tho pcatapeptide chlmomethyl ketone-labeled material under each peak in Fig. 6. Therefore, these ~tsults suggest that the a~tibody recogniz~ ~t struvleral epi~cT~c(s) present in both the ~ c and d.imerlc forms of r P K and which, in order to be detooled, must c4~Utin an ~5¢ntiai aml intact disulfide bond Gnkth8 together ~ catalytic and the regulatory clutins. Whether this ¢pitop¢ is of the discontlnuoe.s type (a portion ol a pepiide ~gmcnt prc.~mt in both chains) or represems a f~.gte linear ix'pride segment cannot he defim~d at this moment. Since Tyr.io6inated or Cys-reduced (or ~tldized) rPK do riot soem to be

recognized by the antiserum, where~-s l_y~];~helcd (by the Bolton Iffuntez zcagent) zPK binds ~'cl[ Io this antibMy, this ~ould sugg~t that the reoogrfized epi~pe~s~ spans a dcunainls) devoid of [] erilicai Ly~ rcsldue~ hut in which one or more Cys a n d / o r Tyr residue~ ,w~m important For immune recognition.

The use of affinity chromatography, in conjunct,on with size ~xehtsjon H P L C in rome cases, re~uhcd in homogen¢ot~ rPK pre~ratlons as ja~sed by N H ztermirml sequea¢,ing. The rat tmzyme was sbo'.vn to have similar NH~-terraini to those repmtnd for human and porci.¢ plazma kalllkrc~n, suggesting Ihc ideatlqal natuzC of these proteins. Thla idrnlificatlon js further supI~atled, hy oampad~on of moT~lur st~JCtUrC,inhibitlon profiles and nature of the cleavage sites as summarized h¢~cin. The a p p a ~ n t molecu]a~ ma&,~c~f the cataLytic chain alone or o:,mplexe,d to th~ r~ulato~ chain r~semhl~ that of plasma kallikrein isofated from different species [5]. Although plasma kallikrain is moSt oftea. ~lerred to as a monomer i l l . it has also b¢~n reported to e×Jst a~ huSh a monomer and a dirncr undo- Certain conditions [18]_ We unsaved both forms using porcine plasma hallikrein (Rg. 7). In the case of rat. the coexistence of both forms of the ~a'myme could partially explain het¢l"ogeneity of the active species obmin~i following size-exclusion HPLC (Fig~. 2 a n d 6). Actually i1 is aot knovm whether a variation in the monomer to dimer ratio oauid have a physiological dgnificance [or the regulatioz] of the ~n~mc activity. In addition to their structural r~cmbla~*,, rhc pro. sent enzyme and plasma kallik~in h a ~ similar catalytic hehaviours. For ~xamplc, both enzyme~ a~¢ ~¢ashlv= to antipaia, leupeptin, ~o~bean trypsin inhibitor bur not to lima be~n trypsit~ inhibitor+ ovomucoid or TLCK (Ref, 7. Table liB). Moruover, the rat enzyme purified in this study and a previous one [7] preferentially cLeave~ peptidcs clth~r C-termlnal to oz in.betweea paired basic residues and, io addition. C.terminal to a basic rcaiduc if preocer~d by o,~,~ o r two hydrophobic amino acids (Table llA). Cleavage preference of this nature has previously l~ecn do0uraentofl in our laboratory wJth a homolosous eneyme obtained for p , ~ i n c and human using a variety of p e p t i d ~ [.5-9,1.1]. loter~stirz$1y, plasma kalhk~in has already ~.,en shown to release bradykinin by cleavage of H M W K in.between a Lys $ Arg pair and C.tcrmlnal to a Phe-ArgL ~qnenoe [19,20]. The p r ~ n ~ of alternate fo~ms of plamna kaIlikr~in in our preparation dese~.~ further comments, First, the rat enzyme preparation oonlained prolein species of smaller mal¢cnlar ma~.~ ( < 83 kD~, Figs. t and 5). ~ - r n b t i a g those r~0orted by other inv~sligamrs [2t,22]. In addition> wc ha,a: and d e l e t e d a NH~-terminal

,*

a?

o

0.,¢

ae

h i L

I

S½

L

J

I

I

I

I._..I

Yig. 7. G~I tiln'athcm ut pol¢ltte l~lilstalil ~,ll~kr~in. Po='Onc plasma ~,a]lik~'i~ 1(I,9 U in c,~¢h ¢'a~.~Iw~-~ in¢lxba[C~JwLLh 12~I-YEFKR-CK f2 ,~M. 3.4 × lIJ'tl0'm/aoPJ~t, 230 r~M ae,A (pH &0) and O.2. ~ m M ED'I-A in a total ~olume at 60 ml. Aner incubation tiE. h, 4=4:'), *.he e,oLtt[~ ~ diiattxl n s addLn~ 124 ml vtth¢ S d fillraliLm ¢l~zficm P,ur/e~ c.nmaim='X~ m~]ecatar mak~ ffro~n mad~¢~ tPt, at~t,aa), Th~ enz)'m¢ so]uUon ~as then ehn:.matu,~-L~pht~i] o~,~l a S~phmJex 13-20D column equUialaltU and~¢[pl,~ with ¢itho" i(Rr~M Mc~ ] m~a EDTA tpH 6.0) ~upp~ pan¢ll ~r I ~ mM ~:~di~mphcv~k~tc, ltO mM NaC1, 1 mM EDTA {nil 8fJt t[~u,xtr [:z~.nelL "1~¢ appa~t~t raol~cul~r mass (Mrl c~f the ~¢patat¢~. r ~iioi= b e,,]¢,,I~el~i~,5 was a¢Ierminod f fauna a ~;alJbra(io=] uulw¢ [[ri~/ll ~bluh'il~ i,l~Jl'~l li~C DC~nl~ma,.~,~r;Jp,h~d ,~lantlllr ,t p i p L¢"JT=5,1 ~ 8[.OW$ fir=Fakir) Jn?'i¢,~t~ the eLutiop ~hi@=]~, ¢~[ Lhe t'RK~D( m I 0.rud thedi~rmr I U l pLa~m/t kaLlikl¢in ~ i c s

c~ved form of plasnm ~llikrein. Miero~quenmng ,:b.ta indicate that a cleavage has o~.un'~d C-ta-mina[ to the ~r.~.u~n.~ Phe~hPhe]2-Arg ]J pn:~at in the ~ l a t o r y chain of th~ enzyme, [t ~s not clear whcthex this cleavage, as yet not re'ported, in the ~iterature, occurred during ~ c pudlicatioa procodures and/m- if it is a physiologically relevant phenomenon calalysed by ;. ::pec~ficenzyme. ]t ~sinreresting to note thai the cleaved form of the tat cnzymc is clcctruphore0cally indlstingui.'.;,hablc from the uncleared c o u n t C ~ r t by

SDS-PAGE analysis (Fig, 2, - $ H , lane 2), This ob~rvatlon we:uM s u ~ s t thal the resulting small peptide encompassing r~idu¢$ 1 to 13 is still linked by s disoifide bond to the ~est of the: regulatory p~ypcptid¢. I n order to deveioF a Sl~-i.t'¢ assay for plasma kalllktein, we produced an emtiserom reacting with the anzytr~ and which could be ssed in RIA. During the pr¢liminsJ'y attempts to astabllsh the R ] A coi'~itions, it was o b ~ r v ~ that antl-rPK antiserum showed a very low ~te~ when we ,J.~d as a ~ the ¢nzyo¢ m dioh0dinated in ti~ presence of hictOperoa/dase. It is pessib~e that an anttgemc deteerninaJ~t of the ergyme bears a tyroslnc res~ae which upon iodlnation v~uld inlerfexe with the |orrnafion of the immune complex. A similar low reactivity with the iodinated ~mzyme was also reported using a mot~oclona] ant/body [23]. We: have: previous~ showed that an antiserum d i ~ against hemaa plasma kallikreln ce.n immunopre~pitate t~s enzyme modified with the aWmity label :=~I. Y E F K R - C K [5]. In this work, .it is showed Chat the enzyme rediolabeled with the affinity reagent can also b¢ used m inca"case the srat,~tivi~ of the RIA. The tow~ limit of deter{ion ol plasarJ~ k~Kkreln in the co~itions of spa: RfA is t~'~ro'~. 3.~ fl~/,~l, a s~tlvi_~ that is approx. 25.fold greater than that reported using an e~-yn'g-~inl~l plasma kaPlkmrt i ~ y in the presrnc:¢ of a monc¢lo~nad antilxxly 123]. The andsrmm developed against the native rPK should be partictdad.y ese:ful in blod~rnica] and iramunolol~cal sthdies of ptasma kallikmin, since it re¢ognizrs the: pro~nase present in crude extracts as w e l l as in puro propa~a6ons. The ros~lts obtained f~om R]A nt~ SDS-PAGE sUtdirs ~ that whe~ rPK is not esposod to ~¢¢larhl8 or' o x i d l z . ~ o o r ~ o a s , the antib o d y I"~o~ni~*~s b o ~ h o]i~cel~fle: f o f n ~ o f the: ¢~,tT,y m e ~n Ol'Kler the: d~"mturl[l~ rxTJ1~itlol~ gsdd ill S D ~ -

forms o f the prol~.nasc, indeed, the 91 kDa on©,¢t'~n zyme:geri form osuld then ~ .tistir~guished from the 91 };Da two-chain ~ctivated from o r the prot=n=~by 5DS-PAGE i"ollowin$ the labchiW, o f its active site with the ch]oromethy[ ketone affinity roagcnt, A.~],s~aedgn~ts We arc indebtr.~d to .tim Rochemont, Nico]a Franco, Odctte ~ q ~ ¢ and A n d r e w Chert for t l ~ r technical cxpertLs¢, The socrctarla) ~ssistance o f Mrs Sy]vle: Emond is a~k=ow]ed~L This investigation was supp~ted b y a fese:asv.h grant from the Mcdlcal gesea~h Council of Canada and by a grant from the United States National Institutes of Health. J.P, and C.L. are supported by the Fonds de la Re=he, the en Sant~' do Qu6hec as a postdoctoral fellow and a '¢he'rehear-boarsi~', respe:ctively. Rtle:~'mtCes z I~ima,k. e_D. end I~,~¢, EW, ttgSl; Me=hods I=r.zymd.SO, 'is7-172, 2 Tans, G,. go~/n~..L,8errettie.LM,, I.lmmle,B, a~d C.~flla,I.H. 3 P~.~I~,F,M.[-I.~.=;ckatup, ['.S'.P.A_and S¢lm]eiamp.:'~.A.~,H. ItgS~ ~. m0LChem.262,7A~l-2477. 4 S~,Io-, J,E., Atlas. S,~, Laragh,2,H.. Sil~tbea'~M, a,~tlKeplaa. A,P,tl~r~ ~r~. Na~LAcad. Sci.USA ~6,.~1t-5918. s S¢idlph,N.O,. I~qein. j.. H~mtcl~z~,L. ~¢'ejatmrL.S. and Chv~ti~, M.(IgBS) ~ (paris) 70, ;)3-46, 6 C~i~ JJ~., S~da~, N.G. an" : t e ~ . n . M . (1~6) ,1, Blot, Chem.Z6t,t0e.~.9-leJ::'o. ~ Seid . g N.G. C~mllsh~ LA. Hamdin. ~'.. Thiha~t. G. and

Ch~er~ M. (195'~'~~Bk~-~,It.~.,o, 83,~=S44. s Seida3L N.C, H¢ady, ,~.b~ H.g~e]in, J,. Paq~in, 2,. I ~ u r ~ C., [~'tlcrJs, ]g-M., P-~Cl¢~li~,J. a l ~ O r ~ M, {tUI~"~ FEBS L¢ll,

211,144-1~0,

9 Mette~s, K.M. R~si~..~,. P ' ~ n ~ J,, (~..~'LL~. M. auad S~idah. ~.G.(Ig08IJ. n~u. -..mm. 263, 12~42-12~53,

PAGE. Since we h a ~ not yet bceo able to srpararo the catalytic and regalatm"~ chains under conditions wkw..h orlty C'~e:a¥CthC ~liiu~l'-tde:~ ]ilLldlt~tile two ¢]~i~1~, of rPK, we cannot asoeftaie, whetlt~ ¢~ther pol)qprptidc can be iedfi-.,Mually xecog, i.,pd b y this po4y¢lona.] antibody+ or wheth~ ~ *JR whole mo~¢~:ule:is ~

I0 Nt~e. H. and ]~at~e~t.j.(1~1t litanies, J, 193.t~?-192. 11 Ctoml~h. ~$A.,~k'F2ah.?,.tS. ~nS C~r,~h¢~.r.t, (1~> J. ~ . 12 LPemmli=tg.E.(l~t Nlt~lg,222,68(I-6B5, 13 g~dal~N.O.,I)tmohm:-gotf©,A-, Lazar¢,(2. A~cgair,F,, Keu.sdL G.3".a ~ Ck~t;e~. M, ( ] ~ , ) J, Bim. C,~m. 2et, ts~zs-to92L t 4 L ~ C~,S~Jt~,,nm~I~.S~Nat~., RJ..Oe]otser~W,~nd ChK-fi¢0,

foc immune recegnittm~ The advantages of RIA as oppougl to activity d~termleatiol~ t,~ng tither synthetic subs~ates or affimty reagents a ~ a specificlty for the stt.~ied proteina~ as welt as the capabiLiG~ to detoc* both the 'o3mogen and ac*,ive [om~ ~d'the Cng'3'L'1~"l'tg,idC~. this m e . t l ~

1~ C~tn~ C'.W~ Fujikawa,K.. McMolI¢o. ~t,A. and Davi¢~EW. t~s~,~) ~cl~missry 2,5,241,D-,:A.IT. ~ , 1~t-1~,. t? Sddah. N,G~ Lndenheim,R~ tn'~l~y, M,. Hamelin, J,, Luffalla, O,, P,~g¢~o, 1% Lam~. C. ~nd Chr~iea. M, 119~) DNA. in

W~qllk[ ] ~ v,'~l'~ tk~fg][ ~ futqfe: f~p~R'iE[l~0t~, dealing with

i s Ce4m,mL R,W,, Mf,ttl~r. L. &~d ~am'z3r,~. (lqgz,9) I. CJir._ tn,.~xt, a&

the heterologoas expt'~slt.~ of rPK eDNA ia v.~ioo~ o~11 lines, F.tars devetcga~nts in our labofatot'y will i n v c l ~ the e • aT this artLis4~nm to isolate tPK f~om ¢om,phix mlxt ...:s by either Woatanop)'eCil~tat~,n m offinity ch,-omatogr~phy. Satoold the~e affmky p r o ~ t ' ~ he ~pccess,fu], it would be po~bl¢ to cbasa~'t¢fiz.¢ the c~talytic state of the socmted a~d intraodlultr storaSe

U-22. tg Taltalteki,¥,, Kitamtwa~N. and NakaaLshi, S (19~9 J. E6ol, Clu:m. 245O,S~z-s~9,

M. t l g ~ ) [i~, J. r,.i,i. P r ~ n

Re.. 3~. 46-aS.

t6 1~(~;,%l),D, and L~un~tT~ W..t. {1977) PJrrJ~. Natl. Ac,ad. ~,2t ~'SA

;~ Tail~ .LF.mul Fuji~aw.& K.~19~.1~. Bi01.C~et~. 25L 1,~)~-t5401.. 2) Koayoemd~an, M,, Btrq~s, D, BL, Mi¢~]a~¢L Y,M., C-oinh~t¢~.

J ~ , Sampal~ C.A,M. and l~pd~. I.L. {]~?l I!~'~, J. Me,t, IDiol, Res,2o. 54~-~.s~,

22 Bed~, Oil. and Ber,.L N. (lqq~l} p~p, B~o~tem. 14. 257-2"/~', Z3 B~dL.G,S. a M ~ N. (IS.°4) Hybfidon',t 3. 2,17-292.