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The inhibitory effect of ω-amino acids and guanidino acids on the activation of α-chymotrypsinogen and trypsinogen
The Inhibitory
Effect of w-Amino
on the Activation
Acids
of Lhymotrypsinogen
and
Guanidino and
Acids
Trypsinogen’
‘I’lte effects of w-:mCn<) neitis and gumidino wicls on tlte :xt,iv:ttion of a-chyn11~ xctrypsinogen ant1 trypsinogen, on the fihrinolytic s!-stern , 2nd 011 tlw protedytic t,ivity of several enzymes htvc been studied. Hotll gr(rups of acids inhibit the rapitl and stow :tct.iv:ttion of a~ct~?llltotrypsinc,gcn; ant1 &:~minovaleric xcitt ant1 -, -gu:tniclinl+ hrtyric acid we the rnc)st effective inhibitors in t,tieir respechve groups. yGii:midin~9butyric :~cid is more pcjtent than &:mlinovaleric acid. ‘he :tutocat:tlytic activnt ion 01 t rypsinogen and tlw activation hy ent erokinasr, previously shown to be m:~rkrtll~ inhibited by d-xninovaleric acid, are blocked even more elf’ectivelg by -ygu:Lnitlinotjutyric acid. Tn contrast to their effect on the :hiv:iticm processes, F-:mrinov;lteric :lcitl ant1 7.gu:lnittinot,ut~ric acid only weakly inhibit t ryptic caseincltysis. TIE inhibition of cll~nloCrypsinc,gen wtiv:tt,ion t)y &aminovnlcric acid antI -, -guani~linohrit\-rib acid is of mixed type, while the inhibitjion of tryptic cnscinotJ-sis is noncompetitive in ctl:lrncter. The inhibition of entcrokimrse tl,v r-gunnitlinot)ut~ri~ xcid is xtso noncorllpetitive. r-C;unnidinot)ut?-ric acid is inferior to e-:tminocapruic acid and &:tminov:deric xicl in inhibiting fibrinolysis. w-.\mino acids arc without effect on the proteot,x-tic zctivit? of a-chymotrypsin, pnp:lin, and pepsin. Gunnidino acids enhance t tla protcolysis t,Jct\yniotrypsin nntt p:tp:tin. &Aminovuleric acid does not, interfere with ttlc xctiwticln of pepsinogen. INTRO
I~T:CxION
Discovery of the inhibitory effect of E-aminocaproic acid on plasmin (1) stimulated considerable interest, ill this amino acid and ill st~ructurally similar compounds. It soon bccamc evident that t-aminocaproic acid was an even more potent inhibitor of plasminogrn activation t.han of plasmin itself (a), and it was also found that several related w-amino acids, differing only in length of the carbon chain, possessed iw Ilibitory activit’y (3, 1). Studies with other ellxyme systems revealed that e-aminocaproic a&l o~rlv slightly decreased the caseii1olytic acti4ty of trypsin (2). But when the influcncc: of w-amilw acids on the nctivatiotl of trypsinogcn \vas invcstigatrd,
they proved to bc potent inhibitors of the autocatalytic activation as well as of the activation by enterokinase, and w-aminocaprylic acid and b-aminovalcric acid jvere even superior to E-aminocaproic acid ill effecCvcness (5). In the light. of these results it, KLS decided to examine t’hc effects of w-amino acids on t,he activation of oc-cllymotrypsinogen, a process which involves trypt,ic hq’drolysi~ of a11 argillyl-isoleucyl linkage (6). .\s rcported below, considerable inhibition was found. When thr kinetic data revealed the inhibition t#o be, ilt part, competit,ive in Ilature, a group of giiatlidillo acid:: was illeluded in t,hc study. ;Zs allalogs of arginine they wcrc expected to hc still mow effective that1 w-amino acids ill inhibititlg chymotrypsinogen activation. This was colifirmcd,
:3’28
(:ICRATZ
aud the findings prompted further extension of the investigation to determine the influe~~ct of guauidino acids on tryptic proteolysis, t’rypsillogeu activation, and plasmit~ogeti activatiou, processes previously show1 to he iuhihited hv w-amino acids. w-:\mino acids aud gualidino acids were also t,ested for the efkct ou several other proteolyt,ic eiizymes. MATERIALS
ASI)
SIETIIOI)S
‘I’rypsin 12X crystallized, salt-free), t,rypsinogen (1X crystallized, 50”;. WIgSO,), a-chymotrypsin (8X crystallized, salt-free), ol-rtls-lnotr?psinogen (OX crystallized, salt-free). enterokinnse (purified). papain (2X crystallized suspension), pepsin (2X crystallized), and pepsinogen were all purcllased fronl Mann Research Lab., Inc. Streptokiuase iynridasej was ol)taincd from Lederle Laf)oratcbries. Tllronlbin (bovine, topiralj was :I product of Parke. l)avis and Co. w-Amino acids, 1,.tyrosine ethyl ester HCI, and ~‘-toluenesutf~~~~~l~ I,-arginine methyl ester I-ICI were also clt)t.ainetl from Mann Research Lat)., Inc. .4ll guanidinll acids wvcre purchased front ttle California Corpor:ltion for Bi~wlrcmical l&rarch. Sodium casrinate powdrr u-as supplied 1)~ Sheffield Chemical.
‘I’lle amounts of chymotrypsin and trypsin formed in the activation nrixt,ures were measured by an esterase assay using as specific substrates TEE? and T,ZME, respertively. ISefore the act,ivntion mixtures were assayed they were diluted to various degrees so that the activity would fall within tile range of measurement. To suppress an?- further activation, buffered 0.1 JI &aminc~valcric acid was used as a diluent, and, for the sanle reason. 6~:llllirrov:lleric acid (0.2 J-1) was included in the assay mixtures. In preliminary expcrinrcnts it was found that &aminovalerir acid in sucll concentrations did not, inhibit t,he TF:l< csterase activity of clrymotrypsin or ttle TA?*lE &erase activity of trypsin. Simi!:trl?-, at a conwntration of 0.1 111. the gu:Lnidino acids and p-:&nine. 1-:rrni]lol)rltvl,i~ acid, and t-aminocaproic acid were without inhibitory effect. Only acid and 6.arninolev~llinic arid w-aniillor:tpryli(, showetl mild intlibiti~,n of the TEE esterase activity of chymotrypsin at a concentration of 0.1 M. However, this did not interfere with ttle 2 The following aI)1)revi:ttions are used: TEE, L-t\-rosinc ethyl ester; TAME, p-toluenesulforl~lL-arginine met.tlyl ester; Tris,tris(hydroxymetllyl) aminomet hane.
determination of chymotrypsin in activation containing those amino acids, because dilution reduced t,he level in the the necessary assay itself to at least 0.01 M.
mixtures
Four-tenths milliliter cjf the act,ivation mixt,ures, dilut)ed as necessary, was added to a mixture of C&12, G-aminovaleric~ acid, and TEE in 0.1 M imidazolc buffer (pH 7.0, 25°C.) to give a total volurrrc of l.(i ml. and final concentrations of 0.1 :I1 CnCly , 0.2 M b-aminovaleric acid, and 0.025 N TEE. The mixtures were inrubated for 15 min. at 25°C.
Four-tenths milliliter of ttle activation nlistures, dilut,ed as necessary, was added to a mixture of G-arninovaleric acid and TAME in 0.1 :II Tris buft’er ipH 7.6, 37°C.) to give a t,otal volume uf 1.6 ml. and final concentrations of 0.2 XI &aminovalerir acid and 0.0163 .I2 TAME. Incubation was for 30 min. at 17°C. < , Hestrin’s colorinletric method as previousI> modified (5) was used to measure the degree of ester hydrolysis in the assay- niixtures. The incnI)ation time with alkaline hydroxylamine solution was 5 min. for the TEE assay and 25 min. for the TALIP; assay. St)sorhancies were read immedately after tjhe addition of FeCls solution.
For the kinetir studies, one TEE unit chymotrypsin or one TMIE unit trypsin was defined as t tie amount of enzyme hydrolyzing one mote ester per minute under the standard conditions of ttle assay. One TEE unit, was contained in 24 pg. cu-~ti~trlot,r~I,sin, and one TAhIIS unit was reprtlsentrd by 5.13 Mg. tt.ypsin.
The caseinolytic method of Kunitz (i) was used with minor modifications. The final substrate concentration was 0.5 g. (‘; if not st,at,ed otherwise. Chynlol rypsin and trypsin werr assayed at pH 8.0 (0.1 ‘11 Tris buffet). I;or papain 0.1 JI ptlosphate buffer at pH 6.8 was employed, and the Assam mixt,ures were fort,ified wit,11 0.001 111Versene and 0.005 M ryst.eine to insure full act,ivity (8). Pepsin activity was determined at pH 1.9.
Ttle St,reptokinase Lysis Time (Y) was found to Ike a ver); convenient method to nwasure the over-all inlilbitory cil’ert, of sut)stnnces on ttlr fibrinolytic system. For great,er reproducibility of
cu-cliymott~ypsitro~ctr l’typsitt cotlwl~ts itit,o ertzymically active a-chymotxypsitr hy cleavage of a sittgle atgittyl-isoleucyl peptide hot~d. .Iut’olytic hrcakdowtt of n-chymotrypsitt thctt leads to folmat’iott of cit,ltcr 6-rlrymot~t~ypsitt or a-chymottypsitt, depettding ott t,he expetGnettta1 cottditiotrs ((j). b-(‘hymottypsitt is ot)taitred if activatiotr is carried out, rapidly with lwgc amouttt,s of \lode of nctibattypsitt, while w4rymott.ypsiti is the pt’etion domitiattt autolytic plodtict if activatioti OCCII~Sslowly itt t.hc presetwe of onlv small amorrtit3 of trypsiti (IO). The cfiect, of Fast 6-Anrinovnteric witI w-amino acids and guatriditto acids OII hot’h w-:~lrlinoc:It)rgtic acid t>ypcs of activatiotr was csamitted. 6-Arninotevulinic acid Iii Table 1 the rwtilbs are summstbx~ (01 E~Atilinoc:tttroic xcitl the six w-amino acids testrd. The order. of &Alaniric itthihitory poteticy was the same f’or twth -, -;Iminot)utyric xicl types of activatiott, nith the csceptiott of b-A4ruinc~v:~lrric acid w-amittocaptylic acid attd c-amittocapwic t~,L\nriIloc:1t)rl)ic :tciti acid wltich escliatige src011tl aitd fottt4i 6-IZrrlinotev~~linic acid place. 6-.2minovalet~ic acid was t,lic sttutigcst~ w-Afliinoc:lt)1,~lic acid itthihitor itt this grortp, nttd it cat] he seeti &Alaninc that t hc ittt,t*oductiott of an osy glottp as itt ;.lilriinc,t)rlt?ric acid 6-ai~iittolevuliriic acid twluccd it,s effectiveIIWS. It is also cvitletrt, that, t>hc potettcy 01 w-amitto acids was ttot, diwct,ly related to for d-amitiovaletic acid attd for Y-gttattidittotnrtyric acid. The slow mode of net ivat,iott t,lrc lcttgth of tjlre cat+ott cltaitt. The tfi’ectivettcss of’ the gttattiditto acids was cltosct~ C’aCI12was omitted from the listed itt ‘l‘ahlr II ittcreased with the chnitt act,ivatiott mistttws itt Utesr studies t)erstts;c: 1cttg;tlt t’t~om guattiditroacet,ic acid to y-g~tu- vat+at,iotts itt tlte C’aC12to cttvrnott..-psitto~t~tt ttiditto\)rrtyt.ic acid. The itthihitoty strength ratio might have itrflucttced the litte:tt.it,y of of the comportttds dcpettdcd also OII the lo- t,lic initial reactiott vclocit,ies. The data 1v0t.c twated accotdittg to I ,itrcwen\.er ;III~ f31rrk c:at.iott 01’ the guattidirto gtwtp 011 it gi\wt aliphatic chaitr attd OIL the pwsetrce or al)- (1 1) altd wrattgrd ott a dottl~lc twipr.ocal settee of addiGotta1 side gtwttps. The pot,ctxy plot. I’igrwc I dcmottstt~atcs that the slopt~s of ~rtattidittopt~opiollic acid, for example, was of itthilritot. atrd cottttx~l valrws (io ttot itttet.. erthattced by shift of the gttattiditto gtur~p sect OII the ordittate as for tlw compctit,ive from the u positiott to tlic tetxiittal /3 loca- t,ype of itrhihitiort, tlor ott tltr abscissa 3s t’iott, and the potettcy of ~~gttattditrot~ut,~~t~i~~for tlic ttottcompetitivc type of ittltil,itiott. acid was decreased by the p~wcttce of att Jttst’ead, they meet itt hctwwtt these tn-t) nmitto gtuttp iii the cr positiott. I~t~oriia conl- axes ittdicatittg tltat itrltihitiott \vas of’ tlte pxisott ot’ the data itt TalAes I alid II it. cair mixed type. The espetimt:ttts wtx~ t,cprat.cltl be sreti t,hat y-gtrattiditiot~tttytic acid was several times t)o ascertaitt the i~eptudtrcibilit~ of t,he wsults. 15ach time the pt~csettcc 01 ;t mow effect.ive thati &amittovalei%~ acid. l’hc kittetics of tlte itrhihit,iott of chynlottottcorz~pc~titiv(~ as well as of :a cottipctitive compottcttt. could IF \-et&d. ttypsittogett :tct,ivatiott \ww iii\-est,igated
330
GERATZ
IKHIBIWRY THE
TABLE EBFECT OF
ACTIVATION
II Acres
C;UASII)INO
OX
OF a-~HPMOTRYPSINOGEN
Composition of activation mixtures and experimental conditions as described in text of Table I. Concentration
Mode ~ of activation
tiuanidino
acid
I
Inhibition, :"n
-Fast
Slow
I y-(:uanidinobutyric ~ acid P-(:uanidinopropionic acid ur,-a~C:uanidinopro~ pionic acid
~~-Gunnidinot)utvric acid i p-C;u:midinopropionic , acid i ulna-C;uanidinopro~ pionic acid C;uanidinoacetic acid L-ol-Amino-6-gu:tnidino-’ valeric acid (nrginine) L-a-.~rnino-y-gu:Initlinobutyric acid
a Inhibitor concentration.
was incoulpletely
%I’ I 01
) -”
65 -7,
~ 88 ~ 67
56
22
0
0
.a$ 0
,
/
,
,
,
.*5
.lO
./5
PO
P.5
,
,
30
35
$S=MG.d-CHYMOTRYPSINOGEN/ML.)
~ 70
42
-a
100 ~ !)7 ~ 83
96 ) 90
1’
47 I 9-l ~ 78 , Gi ] 30
70
-n -I& 22 1 0 i 93 I 76 65 23 ~ 83 70 I 40 19 i
FIG. 1. Plots according to Lineweaver and Burk demonstrating that tile inhibition of the SIOX activation of ol-chrlnotr~psinogen by yguanidinobutgric acid and by &aminovaleric acid is of the mixed type. The cornpositiou of the activation mixtures w-as the same as given in text of Table I except, for t,he omission of CaCle .
I
dissolved
) at this
The inhibition of trypsin by d-aminovaleric acid and y-guanidinobutyric acid was completelv reversible as shown by the following expeiiment. Concentrated solutions of trypsin were incubated for I5 min. with 0.1 N d-aminovaleric acid or 0.03 A1 r-guanidinobutyric acid, then diluted and used in the chymotrypsinogen activation mixtures. When the mixtures were assayed at the end of the activation period, it was found that trypsin pretreated with inhibitor was as effective as an untreated control solution. Simple dilution had reversed all inhibition of the enzyme. INHIBITIOS OF THE CLSEINOI,YTIC ACTIVITY OF TRYFSIK
In view of the results above, the question arose whether the inhibitory substances
would interfere to the same degree with all proteolytic activities of trypsin or whether some processes would be more susceptible to inhibition thau others. To find the answer, the inhibition of chymotrypsinogen activation was compared with the inhibition of tryptic caseinolysis. The two test systems were matched as closely as possible by usiug the same buffer, the same CaCl, colncentration, identical amounts of trypsin, and the same itlcubation time and temperature. The only difference bet8ween the two series was that the activation mixtures contained 14 mg. a-chymot’rypsinogell~l .(i ml. while the caseinolytic mixtures contained 14 mg. casein,! .(i ml. l’igure 2 shows that a-aminovaleric acid and r-guanidinobutyric acid were much more pot’ent inhibitors of chymotrypsinogen activation thaii of the caseinolytic act,icity. The concentration of inhibit01 in the caseinolytic assays had to be IO-100 times greater than in the activation mixtures to achieve the same degree of inhibition. The graph demonstrat,es also that over the mholc range of concent’rations tested r-guanidinobutyric acid was stronger thall &aminovaleric acid ill suppressing activation as well as caseiiiolysis. As seen iii l’ig. 3, t’lie illhibition of caseill proteolysis was noncompetitive in nature. This correlates well with
tltc wsnlts of Alkjaersig cl al. (2) who pre\Gusly found E-aminocaproic acid to l)t: a iiotlcompctiti~t~ itthibitor of the same proc(‘SS.
IGxaust of its cfTecti\-ctirss as ati itihi~~itot of chymot~ypsitiogcti activattioti, y-guaniditiobutyt~ic acid was also tcstcd for its i Iiflt~cttw oii the activatiotl of trypsitiogett stud comparrd with d-aminovalcric acid already kttowt to be a potent inhibitor of the IT~Ctiott (,Y). l’li~ autocatalyt’ic activat,ioti alid t’hc act,ivat,iott I)y ctttctokittase IVCIY~stttdicd. Activation hy e~ltrrokinasc was cart+d ottt itt a slightly acid milieu (pH 6..5) to supp~‘css, at least partially, coticotnitatit~ aittocatalyt,ic activatioti attd the fotmatioti of inert, ptw tciil. ‘J’lit~ picld of activated trypsiiiogvtt ttttder thcw cwttditiotts was ahout G ‘; as compared t’o ahout Xl Y at att alkalitw pII. Iti I’ig. 4 it is clearly drniottstratc~d that, y-guattidilrohtttt,y~i~ acid agaitt outraukwl 6-amiiiovaleric acid as ati itihihitor, tlrc: fotmcr hcittg approximately teii times niorc effective. The kinetics I’ot, tjltc itthihitiott 01 entwokittasc by y~Rttatiitlittot)ittyl.ic :wi(l wcte tltow of a iiottcompetitivt~ rC:wtiotk (Fig. 5).
‘1’1i~assay ~~niploycd iti thew cspcritiic~ttt.s was soiisiti\.t~ to i~ihihitot3 of’ plastnitiogc~tt acti\-at)ioti as well as of plasniiti. Itt ptv 1imiiiat.y t,csts it was fortlid tltat, ~~gitaliidittot)iityrin acid, iii c’otiCt’tit,t.:Lt,iotts as high :LS
CONCENTRATION (Logarlfhmic
OF INHIBITOR Scale)
0
b
F’Ic;. 4. InlCbitory cif’ect ol” r~Ru:lnitlincrl)llt,~~i~ xitl :rntI &:lnlinov:dcric acid on tlw :rutocat xtiv:~t ion of trgpsinc~gcn Co) and c,n Ihe act iv:tt.ion 1)~ (,nteu)kin:m Cb). r\utocat:llyti~ actiwt 0.8 rng. t~rypsinogen, 0.0-l rng. trypsin, Ifi0 prr~c~lcs (:xC12, ant1 inlGhit,or in 1.6 ml. of 0.1 .ll Tris (pH i.6, 4’C.) were incutmted for i Irr. :rt, 4°C. Activ:ltic,n I,y enterokinase: 0.8 ~ng. trypsinogen, cntetwkinmr, and inllil)itor in 1.6 ml. of 0.1 I/ phosplmtc: ))ul’l’er (l)H (i.5, 4°C.) KVL’P incalxltrtl l1r. at 4°C:.
0
I 02
I 04
I 06
I 0.8
I IO
I I2
I 14
I I6
I I.8
alytic ion: I)uR’el 8 mg. for 24
I 2.0
~(S=MG.TRYPSIN~GEN/ML.)
FIG. 5. Plots xcording to I,incwxvrr antI Burli tl(:rllonst.r:LtinK tlutj noncompetitive inllihitor of tl~: activ:ltjicm of trypsinog(rn I)y cntrrokinwe. tnixt uws was as given in text, of Fig. 1.
0.06 AI, did uot delay the format,ion of the filxill clot iii the assav; i.e., thcrc was 110 iuhihitioll of thrombin.“l’hc data in Table III coidirm previous reports (13,4) 011tlic efFectivtutss of c-amilwcapwic acid and A-aminovaleric acid as iiihihitors of fi\willolysis, alld t,hcy also show that r-gual~idiIlo})utylic acid was only a \wak inhibitor iI1 this system.
; -gmniclinol~ut (Zornposition
\-ric acid is :I of :rctiwtion
Iii 0.1 .\I colicelitratioii t,lie w-nmiiio acids Ii&cd in Table I did llot have ally vffect or1 the caseinolytic activity of cu-cttymotrypsiii (:3.X ~(8. c:llzymt/ml.), papain (22 pg;.:‘ml.), or pepsin (17 pg., ml.). :\t the same corlc:~lltl,atiol~, all guallidino acids cllhalwd
pat&dly uttcoilrd prptitlr chains, dlilr cy-c~i~rniot.~ypsillogcll is ptwrtlt in its nati\-r stalk. It is utlktwwtt \vhetlrrt~ w-amitto acids ot’ guaniditto acids play ally physiologic talc us itiltihitow of tlic activattotl of trypsittogrti. rtizytnrs. cliymot,t~ppsitiogri~~ 01’ similw r-~~itatiidit~ohut~t~i~~ acid occttt~s itt the htxiti atld iii ot,ltrt. tissues (I 2), t)rtt8ottly its tirttt~ophysiological rffrct has IKWI st~rtdird (I :I). t-.~iiiitiocapt~oir acid has hrrti ttsctl witlrly iti pat,irtits lvith itict~casrd fibt4tiolytir a(‘tivit,y. Sidr rffrcts which wt’r mow comtiiot~ aftrt. otd t,liati intt*a\-etiotts atlmirtist~t~a~~iotI ittclttded vagur ahtlomitial tlisromfot~t, t~attsra, at~d diatdwt (I-1 11;). III vir\v of’ thr data ptwctttctl otir crt~taittly woti~lrt~s whrtlirt~ itttct~fcwticr \vith thr activntiotr 01 paticwatic rtixytnr ptwittxot~s might, tiot. Itavc cotitt~ihtttrd to t’lir symptoms. chvtnot~ypt~ic plotrolysis /)y applosimnt~ely Hithrt~t~o, it, was impossihlr t,o drtrt~tititw 10“‘; aid pnpaitl ptdrolysis by ahout’ 20 7;. arcttt~ntcly trypsitl itr thr ptwrtw of tt.yp‘Ylrr artivat,iotr of pepxittogetl (2 mg.I’td.) sittogrti 01’ to titrxsut~r rhymott~ypsiti iii t tw at pII I .!I was twt ittflueticcd by 0.1 X ptwrttw of c~iyriiot,t~ypsitto~~~ti and tt-ypsiti, 6-atnitto\.alrt,ir acid. lwrattsc cotitittttitig activat~iott itt t,ltr assa? \\ortltl limit tttr wliahilit,y of tttr twttlth. III G-atiiit~ovalrt~ir ad atid y-gu:~triditio‘J’hr tYw11ts t~rpot~tctl ahovc~ allow 011ly ;L \)ittytG! arid tn-0 agrtlts Iiavr hrrtj f01t1itl spcrulat~ivr atw\vrt~ to the clucstiou why t’hr I\-lticlt will block artivat,iott, atd yrt, will tlot jtihil~i~ioti of ttppsitl should vaty clualitajtitrt++w witlt tlic rstrt,olyt,ir assay of tt,ypsitt tivrly Ivitli a cltatlge of the stthst~t%tc ftY)m atid cltymott~ypsiti. This ahi1it.v will t~~~~t~tcasciti to cllytliott*ypsitlogetl. I’ossihly, the mrtrtl tlic romportt~ds for, ~tsr iti atly system nia~iy lysyl atid wgitiyl gtwtps csposcd iii whrw fttt~thrt~ act,ivat,iott is t,o hr stopprd. cascitl wmprtc so much mow successfully ‘l’l~~y 1na.v IF adtlrti, I’ot. r~atiiplr, to tl~toI’oi, t\ir act.i\-r sit,r of Wpsiti thati thr sit& tlrttsl juice, tight aftrt, it Iliki hrrti o~)taitird, :t\xilnhlc gtwup iii a~cllyrnott.~psittogrtt, that atid activity mrasuwtnrt~ts nil1 their t.rLtlJ thr compctit,ivr compotietrt, thortgh pwsrtltj, tdrrt tjltr amorttits ol’ artivr tqwiti 01’ is 110t rvidctit at, t,hr casc-itt atid itiliil~itot~ rliytnott~ypsiti pwsrtlt. ~otir~~tttt~3tiotis usctl. Thr ptwim~ptiotl wottltl tx that three is ii0 basic ditirtcticc hrtn-rrti the typrs of jttliil)iCott o\wIvrd with the two 1. ,stthst tatrs. .\ltrt~ttat,ively, it could la: as2. sutiirtl that tltc compet,itivr compottct~t is Wily :rlwrtit. I\-it,11caaeiti; i.r., thr act,ive sit,r :i. of tt,ypsitl would /jr rasily accrssildr to cascitr rvrtl itt t,lrt ptYwtw2 of itilti~~ito~, l~ttt .I. w~itltl hc hlorkrd to cllyrttott.ypsittc,gcll. ‘l’hr aflitlit,y f’ot, tltr actix’r site is drtt:txtitwtl tloi 5. otily 1)~ thr aniit~o acid src~urttre of the srtt)sttxtr, bitt also by its molrcttla~ cotifigttt26. tiolr, :uitl it, may t)c ttoted that) rasriti i:. usrd in tltc assays iii it,s tlrunt~tttwl I’oi2n \vit81i 7.
8. KIMMEL,
J.
lt.,
ANI)
Chem. 207, ijl5 9. IATRIDIS,
9.
G.,
a4pp/. I’h?/siol. 10. JACUBSEN,
(“arlsbery,
<:.
SMITH,
E.
J,.,
-1. Hid.
(1951). ANI)
~ERGCSON,
J. H.,
.I.
18, 387 (1%X3). F.,
('07ilpt.
/l'c?W/.
7'1X2'.
kh.
SEr. (Ihim. 26, 325 (194i).
H., AXI) ~<~.RI<, I>., J. .I,?/. Chem. Sot. 66, 65X (1934). 12. IRREVERRX, I'., EVANS, I<. I,., HAYI)PS, A. R., ASI) PILBER, J<., .~‘ulirrc! 180, 701 (1957). 11. J.ISEWE.4VER,
13. I'IJRPI:I~A, I). I’., G~RADO hl., ASI) GKT SU~~WF, H., Science 127, 1170 (1958). I,+. NILSSON, I. hI., JklijRKMAN, s. Ji:., AS,) AX170, 487 I)ERSSOx, J,., Llcta wild. Scarrtl. (19x). 15. kk'~IC'Ol,, SIG, S.,
(l%il).
(;. I'., J~LET(!IlER, 9. ,491) SHERRY, S., .I.
1'. iZLP;.JAERC7d. 86, 829
Effect of w-Amino
on the Activation
Acids
of Lhymotrypsinogen
and
Guanidino and
Acids
Trypsinogen’
‘I’lte effects of w-:mCn<) neitis and gumidino wicls on tlte :xt,iv:ttion of a-chyn11~ xctrypsinogen ant1 trypsinogen, on the fihrinolytic s!-stern , 2nd 011 tlw protedytic t,ivity of several enzymes htvc been studied. Hotll gr(rups of acids inhibit the rapitl and stow :tct.iv:ttion of a~ct~?llltotrypsinc,gcn; ant1 &:~minovaleric xcitt ant1 -, -gu:tniclinl+ hrtyric acid we the rnc)st effective inhibitors in t,tieir respechve groups. yGii:midin~9butyric :~cid is more pcjtent than &:mlinovaleric acid. ‘he :tutocat:tlytic activnt ion 01 t rypsinogen and tlw activation hy ent erokinasr, previously shown to be m:~rkrtll~ inhibited by d-xninovaleric acid, are blocked even more elf’ectivelg by -ygu:Lnitlinotjutyric acid. Tn contrast to their effect on the :hiv:iticm processes, F-:mrinov;lteric :lcitl ant1 7.gu:lnittinot,ut~ric acid only weakly inhibit t ryptic caseincltysis. TIE inhibition of cll~nloCrypsinc,gen wtiv:tt,ion t)y &aminovnlcric acid antI -, -guani~linohrit\-rib acid is of mixed type, while the inhibitjion of tryptic cnscinotJ-sis is noncompetitive in ctl:lrncter. The inhibition of entcrokimrse tl,v r-gunnitlinot)ut~ri~ xcid is xtso noncorllpetitive. r-C;unnidinot)ut?-ric acid is inferior to e-:tminocapruic acid and &:tminov:deric xicl in inhibiting fibrinolysis. w-.\mino acids arc without effect on the proteot,x-tic zctivit? of a-chymotrypsin, pnp:lin, and pepsin. Gunnidino acids enhance t tla protcolysis t,Jct\yniotrypsin nntt p:tp:tin. &Aminovuleric acid does not, interfere with ttlc xctiwticln of pepsinogen. INTRO
I~T:CxION
Discovery of the inhibitory effect of E-aminocaproic acid on plasmin (1) stimulated considerable interest, ill this amino acid and ill st~ructurally similar compounds. It soon bccamc evident that t-aminocaproic acid was an even more potent inhibitor of plasminogrn activation t.han of plasmin itself (a), and it was also found that several related w-amino acids, differing only in length of the carbon chain, possessed iw Ilibitory activit’y (3, 1). Studies with other ellxyme systems revealed that e-aminocaproic a&l o~rlv slightly decreased the caseii1olytic acti4ty of trypsin (2). But when the influcncc: of w-amilw acids on the nctivatiotl of trypsinogcn \vas invcstigatrd,
they proved to bc potent inhibitors of the autocatalytic activation as well as of the activation by enterokinase, and w-aminocaprylic acid and b-aminovalcric acid jvere even superior to E-aminocaproic acid ill effecCvcness (5). In the light. of these results it, KLS decided to examine t’hc effects of w-amino acids on t,he activation of oc-cllymotrypsinogen, a process which involves trypt,ic hq’drolysi~ of a11 argillyl-isoleucyl linkage (6). .\s rcported below, considerable inhibition was found. When thr kinetic data revealed the inhibition t#o be, ilt part, competit,ive in Ilature, a group of giiatlidillo acid:: was illeluded in t,hc study. ;Zs allalogs of arginine they wcrc expected to hc still mow effective that1 w-amino acids ill inhibititlg chymotrypsinogen activation. This was colifirmcd,
:3’28
(:ICRATZ
aud the findings prompted further extension of the investigation to determine the influe~~ct of guauidino acids on tryptic proteolysis, t’rypsillogeu activation, and plasmit~ogeti activatiou, processes previously show1 to he iuhihited hv w-amino acids. w-:\mino acids aud gualidino acids were also t,ested for the efkct ou several other proteolyt,ic eiizymes. MATERIALS
ASI)
SIETIIOI)S
‘I’rypsin 12X crystallized, salt-free), t,rypsinogen (1X crystallized, 50”;. WIgSO,), a-chymotrypsin (8X crystallized, salt-free), ol-rtls-lnotr?psinogen (OX crystallized, salt-free). enterokinnse (purified). papain (2X crystallized suspension), pepsin (2X crystallized), and pepsinogen were all purcllased fronl Mann Research Lab., Inc. Streptokiuase iynridasej was ol)taincd from Lederle Laf)oratcbries. Tllronlbin (bovine, topiralj was :I product of Parke. l)avis and Co. w-Amino acids, 1,.tyrosine ethyl ester HCI, and ~‘-toluenesutf~~~~~l~ I,-arginine methyl ester I-ICI were also clt)t.ainetl from Mann Research Lat)., Inc. .4ll guanidinll acids wvcre purchased front ttle California Corpor:ltion for Bi~wlrcmical l&rarch. Sodium casrinate powdrr u-as supplied 1)~ Sheffield Chemical.
‘I’lle amounts of chymotrypsin and trypsin formed in the activation nrixt,ures were measured by an esterase assay using as specific substrates TEE? and T,ZME, respertively. ISefore the act,ivntion mixtures were assayed they were diluted to various degrees so that the activity would fall within tile range of measurement. To suppress an?- further activation, buffered 0.1 JI &aminc~valcric acid was used as a diluent, and, for the sanle reason. 6~:llllirrov:lleric acid (0.2 J-1) was included in the assay mixtures. In preliminary expcrinrcnts it was found that &aminovalerir acid in sucll concentrations did not, inhibit t,he TF:l< csterase activity of clrymotrypsin or ttle TA?*lE &erase activity of trypsin. Simi!:trl?-, at a conwntration of 0.1 111. the gu:Lnidino acids and p-:&nine. 1-:rrni]lol)rltvl,i~ acid, and t-aminocaproic acid were without inhibitory effect. Only acid and 6.arninolev~llinic arid w-aniillor:tpryli(, showetl mild intlibiti~,n of the TEE esterase activity of chymotrypsin at a concentration of 0.1 M. However, this did not interfere with ttle 2 The following aI)1)revi:ttions are used: TEE, L-t\-rosinc ethyl ester; TAME, p-toluenesulforl~lL-arginine met.tlyl ester; Tris,tris(hydroxymetllyl) aminomet hane.
determination of chymotrypsin in activation containing those amino acids, because dilution reduced t,he level in the the necessary assay itself to at least 0.01 M.
mixtures
Four-tenths milliliter cjf the act,ivation mixt,ures, dilut)ed as necessary, was added to a mixture of C&12, G-aminovaleric~ acid, and TEE in 0.1 M imidazolc buffer (pH 7.0, 25°C.) to give a total volurrrc of l.(i ml. and final concentrations of 0.1 :I1 CnCly , 0.2 M b-aminovaleric acid, and 0.025 N TEE. The mixtures were inrubated for 15 min. at 25°C.
Four-tenths milliliter of ttle activation nlistures, dilut,ed as necessary, was added to a mixture of G-arninovaleric acid and TAME in 0.1 :II Tris buft’er ipH 7.6, 37°C.) to give a t,otal volume uf 1.6 ml. and final concentrations of 0.2 XI &aminovalerir acid and 0.0163 .I2 TAME. Incubation was for 30 min. at 17°C. < , Hestrin’s colorinletric method as previousI> modified (5) was used to measure the degree of ester hydrolysis in the assay- niixtures. The incnI)ation time with alkaline hydroxylamine solution was 5 min. for the TEE assay and 25 min. for the TALIP; assay. St)sorhancies were read immedately after tjhe addition of FeCls solution.
For the kinetir studies, one TEE unit chymotrypsin or one TMIE unit trypsin was defined as t tie amount of enzyme hydrolyzing one mote ester per minute under the standard conditions of ttle assay. One TEE unit, was contained in 24 pg. cu-~ti~trlot,r~I,sin, and one TAhIIS unit was reprtlsentrd by 5.13 Mg. tt.ypsin.
The caseinolytic method of Kunitz (i) was used with minor modifications. The final substrate concentration was 0.5 g. (‘; if not st,at,ed otherwise. Chynlol rypsin and trypsin werr assayed at pH 8.0 (0.1 ‘11 Tris buffet). I;or papain 0.1 JI ptlosphate buffer at pH 6.8 was employed, and the Assam mixt,ures were fort,ified wit,11 0.001 111Versene and 0.005 M ryst.eine to insure full act,ivity (8). Pepsin activity was determined at pH 1.9.
Ttle St,reptokinase Lysis Time (Y) was found to Ike a ver); convenient method to nwasure the over-all inlilbitory cil’ert, of sut)stnnces on ttlr fibrinolytic system. For great,er reproducibility of
cu-cliymott~ypsitro~ctr l’typsitt cotlwl~ts itit,o ertzymically active a-chymotxypsitr hy cleavage of a sittgle atgittyl-isoleucyl peptide hot~d. .Iut’olytic hrcakdowtt of n-chymotrypsitt thctt leads to folmat’iott of cit,ltcr 6-rlrymot~t~ypsitt or a-chymottypsitt, depettding ott t,he expetGnettta1 cottditiotrs ((j). b-(‘hymottypsitt is ot)taitred if activatiotr is carried out, rapidly with lwgc amouttt,s of \lode of nctibattypsitt, while w4rymott.ypsiti is the pt’etion domitiattt autolytic plodtict if activatioti OCCII~Sslowly itt t.hc presetwe of onlv small amorrtit3 of trypsiti (IO). The cfiect, of Fast 6-Anrinovnteric witI w-amino acids and guatriditto acids OII hot’h w-:~lrlinoc:It)rgtic acid t>ypcs of activatiotr was csamitted. 6-Arninotevulinic acid Iii Table 1 the rwtilbs are summstbx~ (01 E~Atilinoc:tttroic xcitl the six w-amino acids testrd. The order. of &Alaniric itthihitory poteticy was the same f’or twth -, -;Iminot)utyric xicl types of activatiott, nith the csceptiott of b-A4ruinc~v:~lrric acid w-amittocaptylic acid attd c-amittocapwic t~,L\nriIloc:1t)rl)ic :tciti acid wltich escliatige src011tl aitd fottt4i 6-IZrrlinotev~~linic acid place. 6-.2minovalet~ic acid was t,lic sttutigcst~ w-Afliinoc:lt)1,~lic acid itthihitor itt this grortp, nttd it cat] he seeti &Alaninc that t hc ittt,t*oductiott of an osy glottp as itt ;.lilriinc,t)rlt?ric acid 6-ai~iittolevuliriic acid twluccd it,s effectiveIIWS. It is also cvitletrt, that, t>hc potettcy 01 w-amitto acids was ttot, diwct,ly related to for d-amitiovaletic acid attd for Y-gttattidittotnrtyric acid. The slow mode of net ivat,iott t,lrc lcttgth of tjlre cat+ott cltaitt. The tfi’ectivettcss of’ the gttattiditto acids was cltosct~ C’aCI12was omitted from the listed itt ‘l‘ahlr II ittcreased with the chnitt act,ivatiott mistttws itt Utesr studies t)erstts;c: 1cttg;tlt t’t~om guattiditroacet,ic acid to y-g~tu- vat+at,iotts itt tlte C’aC12to cttvrnott..-psitto~t~tt ttiditto\)rrtyt.ic acid. The itthihitoty strength ratio might have itrflucttced the litte:tt.it,y of of the comportttds dcpettdcd also OII the lo- t,lic initial reactiott vclocit,ies. The data 1v0t.c twated accotdittg to I ,itrcwen\.er ;III~ f31rrk c:at.iott 01’ the guattidirto gtwtp 011 it gi\wt aliphatic chaitr attd OIL the pwsetrce or al)- (1 1) altd wrattgrd ott a dottl~lc twipr.ocal settee of addiGotta1 side gtwttps. The pot,ctxy plot. I’igrwc I dcmottstt~atcs that the slopt~s of ~rtattidittopt~opiollic acid, for example, was of itthilritot. atrd cottttx~l valrws (io ttot itttet.. erthattced by shift of the gttattiditto gtur~p sect OII the ordittate as for tlw compctit,ive from the u positiott to tlic tetxiittal /3 loca- t,ype of itrhihitiort, tlor ott tltr abscissa 3s t’iott, and the potettcy of ~~gttattditrot~ut,~~t~i~~for tlic ttottcompetitivc type of ittltil,itiott. acid was decreased by the p~wcttce of att Jttst’ead, they meet itt hctwwtt these tn-t) nmitto gtuttp iii the cr positiott. I~t~oriia conl- axes ittdicatittg tltat itrltihitiott \vas of’ tlte pxisott ot’ the data itt TalAes I alid II it. cair mixed type. The espetimt:ttts wtx~ t,cprat.cltl be sreti t,hat y-gtrattiditiot~tttytic acid was several times t)o ascertaitt the i~eptudtrcibilit~ of t,he wsults. 15ach time the pt~csettcc 01 ;t mow effect.ive thati &amittovalei%~ acid. l’hc kittetics of tlte itrhihit,iott of chynlottottcorz~pc~titiv(~ as well as of :a cottipctitive compottcttt. could IF \-et&d. ttypsittogett :tct,ivatiott \ww iii\-est,igated
330
GERATZ
IKHIBIWRY THE
TABLE EBFECT OF
ACTIVATION
II Acres
C;UASII)INO
OX
OF a-~HPMOTRYPSINOGEN
Composition of activation mixtures and experimental conditions as described in text of Table I. Concentration
Mode ~ of activation
tiuanidino
acid
I
Inhibition, :"n
-Fast
Slow
I y-(:uanidinobutyric ~ acid P-(:uanidinopropionic acid ur,-a~C:uanidinopro~ pionic acid
~~-Gunnidinot)utvric acid i p-C;u:midinopropionic , acid i ulna-C;uanidinopro~ pionic acid C;uanidinoacetic acid L-ol-Amino-6-gu:tnidino-’ valeric acid (nrginine) L-a-.~rnino-y-gu:Initlinobutyric acid
a Inhibitor concentration.
was incoulpletely
%I’ I 01
) -”
65 -7,
~ 88 ~ 67
56
22
0
0
.a$ 0
,
/
,
,
,
.*5
.lO
./5
PO
P.5
,
,
30
35
$S=MG.d-CHYMOTRYPSINOGEN/ML.)
~ 70
42
-a
100 ~ !)7 ~ 83
96 ) 90
1’
47 I 9-l ~ 78 , Gi ] 30
70
-n -I& 22 1 0 i 93 I 76 65 23 ~ 83 70 I 40 19 i
FIG. 1. Plots according to Lineweaver and Burk demonstrating that tile inhibition of the SIOX activation of ol-chrlnotr~psinogen by yguanidinobutgric acid and by &aminovaleric acid is of the mixed type. The cornpositiou of the activation mixtures w-as the same as given in text of Table I except, for t,he omission of CaCle .
I
dissolved
) at this
The inhibition of trypsin by d-aminovaleric acid and y-guanidinobutyric acid was completelv reversible as shown by the following expeiiment. Concentrated solutions of trypsin were incubated for I5 min. with 0.1 N d-aminovaleric acid or 0.03 A1 r-guanidinobutyric acid, then diluted and used in the chymotrypsinogen activation mixtures. When the mixtures were assayed at the end of the activation period, it was found that trypsin pretreated with inhibitor was as effective as an untreated control solution. Simple dilution had reversed all inhibition of the enzyme. INHIBITIOS OF THE CLSEINOI,YTIC ACTIVITY OF TRYFSIK
In view of the results above, the question arose whether the inhibitory substances
would interfere to the same degree with all proteolytic activities of trypsin or whether some processes would be more susceptible to inhibition thau others. To find the answer, the inhibition of chymotrypsinogen activation was compared with the inhibition of tryptic caseinolysis. The two test systems were matched as closely as possible by usiug the same buffer, the same CaCl, colncentration, identical amounts of trypsin, and the same itlcubation time and temperature. The only difference bet8ween the two series was that the activation mixtures contained 14 mg. a-chymot’rypsinogell~l .(i ml. while the caseinolytic mixtures contained 14 mg. casein,! .(i ml. l’igure 2 shows that a-aminovaleric acid and r-guanidinobutyric acid were much more pot’ent inhibitors of chymotrypsinogen activation thaii of the caseinolytic act,icity. The concentration of inhibit01 in the caseinolytic assays had to be IO-100 times greater than in the activation mixtures to achieve the same degree of inhibition. The graph demonstrat,es also that over the mholc range of concent’rations tested r-guanidinobutyric acid was stronger thall &aminovaleric acid ill suppressing activation as well as caseiiiolysis. As seen iii l’ig. 3, t’lie illhibition of caseill proteolysis was noncompetitive in nature. This correlates well with
tltc wsnlts of Alkjaersig cl al. (2) who pre\Gusly found E-aminocaproic acid to l)t: a iiotlcompctiti~t~ itthibitor of the same proc(‘SS.
IGxaust of its cfTecti\-ctirss as ati itihi~~itot of chymot~ypsitiogcti activattioti, y-guaniditiobutyt~ic acid was also tcstcd for its i Iiflt~cttw oii the activatiotl of trypsitiogett stud comparrd with d-aminovalcric acid already kttowt to be a potent inhibitor of the IT~Ctiott (,Y). l’li~ autocatalyt’ic activat,ioti alid t’hc act,ivat,iott I)y ctttctokittase IVCIY~stttdicd. Activation hy e~ltrrokinasc was cart+d ottt itt a slightly acid milieu (pH 6..5) to supp~‘css, at least partially, coticotnitatit~ aittocatalyt,ic activatioti attd the fotmatioti of inert, ptw tciil. ‘J’lit~ picld of activated trypsiiiogvtt ttttder thcw cwttditiotts was ahout G ‘; as compared t’o ahout Xl Y at att alkalitw pII. Iti I’ig. 4 it is clearly drniottstratc~d that, y-guattidilrohtttt,y~i~ acid agaitt outraukwl 6-amiiiovaleric acid as ati itihihitor, tlrc: fotmcr hcittg approximately teii times niorc effective. The kinetics I’ot, tjltc itthihitiott 01 entwokittasc by y~Rttatiitlittot)ittyl.ic :wi(l wcte tltow of a iiottcompetitivt~ rC:wtiotk (Fig. 5).
‘1’1i~assay ~~niploycd iti thew cspcritiic~ttt.s was soiisiti\.t~ to i~ihihitot3 of’ plastnitiogc~tt acti\-at)ioti as well as of plasniiti. Itt ptv 1imiiiat.y t,csts it was fortlid tltat, ~~gitaliidittot)iityrin acid, iii c’otiCt’tit,t.:Lt,iotts as high :LS
CONCENTRATION (Logarlfhmic
OF INHIBITOR Scale)
0
b
F’Ic;. 4. InlCbitory cif’ect ol” r~Ru:lnitlincrl)llt,~~i~ xitl :rntI &:lnlinov:dcric acid on tlw :rutocat xtiv:~t ion of trgpsinc~gcn Co) and c,n Ihe act iv:tt.ion 1)~ (,nteu)kin:m Cb). r\utocat:llyti~ actiwt 0.8 rng. t~rypsinogen, 0.0-l rng. trypsin, Ifi0 prr~c~lcs (:xC12, ant1 inlGhit,or in 1.6 ml. of 0.1 .ll Tris (pH i.6, 4’C.) were incutmted for i Irr. :rt, 4°C. Activ:ltic,n I,y enterokinase: 0.8 ~ng. trypsinogen, cntetwkinmr, and inllil)itor in 1.6 ml. of 0.1 I/ phosplmtc: ))ul’l’er (l)H (i.5, 4°C.) KVL’P incalxltrtl l1r. at 4°C:.
0
I 02
I 04
I 06
I 0.8
I IO
I I2
I 14
I I6
I I.8
alytic ion: I)uR’el 8 mg. for 24
I 2.0
~(S=MG.TRYPSIN~GEN/ML.)
FIG. 5. Plots xcording to I,incwxvrr antI Burli tl(:rllonst.r:LtinK tlutj noncompetitive inllihitor of tl~: activ:ltjicm of trypsinog(rn I)y cntrrokinwe. tnixt uws was as given in text, of Fig. 1.
0.06 AI, did uot delay the format,ion of the filxill clot iii the assav; i.e., thcrc was 110 iuhihitioll of thrombin.“l’hc data in Table III coidirm previous reports (13,4) 011tlic efFectivtutss of c-amilwcapwic acid and A-aminovaleric acid as iiihihitors of fi\willolysis, alld t,hcy also show that r-gual~idiIlo})utylic acid was only a \wak inhibitor iI1 this system.
; -gmniclinol~ut (Zornposition
\-ric acid is :I of :rctiwtion
Iii 0.1 .\I colicelitratioii t,lie w-nmiiio acids Ii&cd in Table I did llot have ally vffect or1 the caseinolytic activity of cu-cttymotrypsiii (:3.X ~(8. c:llzymt/ml.), papain (22 pg;.:‘ml.), or pepsin (17 pg., ml.). :\t the same corlc:~lltl,atiol~, all guallidino acids cllhalwd
pat&dly uttcoilrd prptitlr chains, dlilr cy-c~i~rniot.~ypsillogcll is ptwrtlt in its nati\-r stalk. It is utlktwwtt \vhetlrrt~ w-amitto acids ot’ guaniditto acids play ally physiologic talc us itiltihitow of tlic activattotl of trypsittogrti. rtizytnrs. cliymot,t~ppsitiogri~~ 01’ similw r-~~itatiidit~ohut~t~i~~ acid occttt~s itt the htxiti atld iii ot,ltrt. tissues (I 2), t)rtt8ottly its tirttt~ophysiological rffrct has IKWI st~rtdird (I :I). t-.~iiiitiocapt~oir acid has hrrti ttsctl witlrly iti pat,irtits lvith itict~casrd fibt4tiolytir a(‘tivit,y. Sidr rffrcts which wt’r mow comtiiot~ aftrt. otd t,liati intt*a\-etiotts atlmirtist~t~a~~iotI ittclttded vagur ahtlomitial tlisromfot~t, t~attsra, at~d diatdwt (I-1 11;). III vir\v of’ thr data ptwctttctl otir crt~taittly woti~lrt~s whrtlirt~ itttct~fcwticr \vith thr activntiotr 01 paticwatic rtixytnr ptwittxot~s might, tiot. Itavc cotitt~ihtttrd to t’lir symptoms. chvtnot~ypt~ic plotrolysis /)y applosimnt~ely Hithrt~t~o, it, was impossihlr t,o drtrt~tititw 10“‘; aid pnpaitl ptdrolysis by ahout’ 20 7;. arcttt~ntcly trypsitl itr thr ptwrtw of tt.yp‘Ylrr artivat,iotr of pepxittogetl (2 mg.I’td.) sittogrti 01’ to titrxsut~r rhymott~ypsiti iii t tw at pII I .!I was twt ittflueticcd by 0.1 X ptwrttw of c~iyriiot,t~ypsitto~~~ti and tt-ypsiti, 6-atnitto\.alrt,ir acid. lwrattsc cotitittttitig activat~iott itt t,ltr assa? \\ortltl limit tttr wliahilit,y of tttr twttlth. III G-atiiit~ovalrt~ir ad atid y-gu:~triditio‘J’hr tYw11ts t~rpot~tctl ahovc~ allow 011ly ;L \)ittytG! arid tn-0 agrtlts Iiavr hrrtj f01t1itl spcrulat~ivr atw\vrt~ to the clucstiou why t’hr I\-lticlt will block artivat,iott, atd yrt, will tlot jtihil~i~ioti of ttppsitl should vaty clualitajtitrt++w witlt tlic rstrt,olyt,ir assay of tt,ypsitt tivrly Ivitli a cltatlge of the stthst~t%tc ftY)m atid cltymott~ypsiti. This ahi1it.v will t~~~~t~tcasciti to cllytliott*ypsitlogetl. I’ossihly, the mrtrtl tlic romportt~ds for, ~tsr iti atly system nia~iy lysyl atid wgitiyl gtwtps csposcd iii whrw fttt~thrt~ act,ivat,iott is t,o hr stopprd. cascitl wmprtc so much mow successfully ‘l’l~~y 1na.v IF adtlrti, I’ot. r~atiiplr, to tl~toI’oi, t\ir act.i\-r sit,r of Wpsiti thati thr sit& tlrttsl juice, tight aftrt, it Iliki hrrti o~)taitird, :t\xilnhlc gtwup iii a~cllyrnott.~psittogrtt, that atid activity mrasuwtnrt~ts nil1 their t.rLtlJ thr compctit,ivr compotietrt, thortgh pwsrtltj, tdrrt tjltr amorttits ol’ artivr tqwiti 01’ is 110t rvidctit at, t,hr casc-itt atid itiliil~itot~ rliytnott~ypsiti pwsrtlt. ~otir~~tttt~3tiotis usctl. Thr ptwim~ptiotl wottltl tx that three is ii0 basic ditirtcticc hrtn-rrti the typrs of jttliil)iCott o\wIvrd with the two 1. ,stthst tatrs. .\ltrt~ttat,ively, it could la: as2. sutiirtl that tltc compet,itivr compottct~t is Wily :rlwrtit. I\-it,11caaeiti; i.r., thr act,ive sit,r :i. of tt,ypsitl would /jr rasily accrssildr to cascitr rvrtl itt t,lrt ptYwtw2 of itilti~~ito~, l~ttt .I. w~itltl hc hlorkrd to cllyrttott.ypsittc,gcll. ‘l’hr aflitlit,y f’ot, tltr actix’r site is drtt:txtitwtl tloi 5. otily 1)~ thr aniit~o acid src~urttre of the srtt)sttxtr, bitt also by its molrcttla~ cotifigttt26. tiolr, :uitl it, may t)c ttoted that) rasriti i:. usrd in tltc assays iii it,s tlrunt~tttwl I’oi2n \vit81i 7.
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