VOL. 17 (1957) STUDIES IN POLAROGRAPHIC ANALYSIS. I. 439

Lc complcxc manganbsc( I I I)-tn6thanolamtnc. obtenu par oxldatton du mangan&se(II) par I’oxyg&nc dc l’alr. cn prCsence d’hydroxydc dc sodium o.5M ct de tr&thanolammc h 4%. pcut i?tro utths6 pour lc dosage polarographlquc du manganCsc Dans lcs condttlons obscrvbes, IO courant de dlffuslon vane 1mCalrcmcnt pour des concentrations en mnngadso allant de 2 x0-4 h 7 IO-~M La valcur En/. est dgalc P -0 5 V (vs EC S ) Lc cocfflclcnt dc temperature du courant dc dlffuslon est dc I z”A, par dcgrb Lcs valcurs dc I ct Do dlmlnuent en fonctlon dc l’augmcntatron dcb conccn- tratlons cn hydroxydc alcahn ct cn trlCthanolamlnc En pr&scnce do NaOH o 5M ot dc 4% do tr&thanolammc, I = I 27 ct Do - 4 I 8.10-o cm’/scc.

%USA,MMENFASSUNG

Der .Mangan(III)komplcx mlt TrtYthanolamin dcr durch Oxydatlon elnor Mangan(II)lOsung m Anwcsenhclt von o 5M NnOH und 4 Volumprozcnt TrlYthanolamrn mlt Luftsaucrstoff hcrgcstcllt wlrd, hat such fdr dlc polarographlschc Bestlmmung dcs Mangans gut bcw8hrt Dcr Dlffuslonstrom 1st dcr Mangankonrontratlon von 2.10-4 bls 7. IO -JM proportional. FUr the We110 Mn+‘/Mn+* Irt

Z:Iirn 1st 1.2O’

= -0 5 V gcgcn due gcsYtttgtc Kalomclcktrodc Dcr Tcmpcraturkocfflzlcnt fBr den Diffusion- ,(, pro Grad I und D nchmcn abwcnn dlc Alkali- und dlc TrlYthnnolnmlnkonrcntratlon

tunchmcn. Sic betragcn in Anwcscnhclt von o 5M XaOH uncl 4oA, Trltithnnolamtn I = I 27 und Do 0 4 189 IO-. cm’/scc

REFERENCES

E. HA~~AMOTO. Colfecrro~r CrcchosCov. Chrm Com~u~s , 6 (1938) 3 I 5 J Piu~ste~, Cofkclron Crechoslov. Chem Commrtns , 3 (1931) 406 >I VOHISKVA. Collcclron Crcchostov. C/rem Conrtnwu.. x I (1939) 588 E T VP.RD~ER.CO~~CC~~OI~ C:ecI~oslov. Chews Commtcns , II (1939)216 E T. VERDIER. Collrcf~on C:cchoslov. Chem Communs , I I (x939) 233

a I >I KOLTIEOYY ANV J I \VATTSHS, Ind fZ)~g Citenl , Anal Ed , 15 (1943) 8 a J MoJ%, PYOC fulern Polarogr. Congress, Prague. 1951, Pt I(r951) 638. Pt I11 (1952) 464 ‘ J \’ A NOVAK, J 1Cvr~ ASD J Rt,tA. Cltem f~sly. 47 (1953) 649. s J Rrttrr AND L SP.RAK, ColIecr;on Cteci~oslov. Cltent Commctns , 20 (1955) 640 l I >I. ISSA AND I I: HEWA:DY. Chem:sl A9SU@Sf. 55 (1955) 70 ’ bf GIUAUD. Compf rend.. 236 (1953) 814 l I M ISSA, S E KHALAPALLA AND R >I ISSA. Ret /rav ch:nr , 75 (rq5G) 1031 . L ~~IXTES, PoIarograph:c Techn~qucs. Intcrsclcncc Pubhshcm. Izmdon. 1956. p 56

Rccc~vcd October qth, 1956

RESOLUTION OF CRYSTALLINE RIBONUCLEASE BY PAPER ELECTROPHORESIS

bY

ANWAR A HAKIM

Departmtnl of Mrdwal Research. Nattonal Chcldren’s Cardrac Hospalal. Msam8. i;(o (U s A )

INTRODUCTIOS

Modifications and refinements in the techmques of paper electrophoresls may in- crease the efficacy of this technique for fractionating proteins beyond its present value The present report 1s an elaboration of effects on the resolution of ribonuclease frac- tions on paper produced by two-dimensi~nal~~~lectrophorcsis. The aim of this proce- dure is to provide a more accurate analytical stgndard of Fnzyfnc punty by dcmon- stratina oossiblc hctcrovcnertv of crvstalhne entvmc. which was reported1 to be homo-

440 A. A. HAKISI VOL. 17 (1957)

gencous when rt was studtcd m the Trselius electrophoresrs. Crystalhnc rrbonuclease (RSase) has been shown to be rcsolvablc into two components by partrtron*, as well as by ion exchange resm3 chromatography.

The present mvcstrgattons with paper clectrophorcsrr revealed for the first trme the analyttcal resolutron capacity of two-drmensronal paper clcetrophorcsrs Crystailme rrbonuclcase consisted of at least four enr.ymically acttvc components resolvable by paper elcctrophoresrs. The spccrflc enzymlc actrvity of each of the fractions is demon- strateti.

EXPERIblENTAL

CytlIlInc-z’,3’-plloaphatc and .ictcnc;iiIIlc-z’.3’-ptlosptrntc wcrc xynthcstzcti chcmIc~liy by the mCthOd Of 13HOWN. MMACRATH AND ‘1ODn’

GuaIIosInc-2’.3’-phosphate was Isolutcd by a prcvIoI:sly drscrIbcc1 pro~ctlurc*. a\ modIfIed by f-izr*f*m-. Wtimw~r~ AND MA~~KEIAM*

fjrIclInc-2’,3’-i~horil~l~,tte was Isolated from RNnvc dIKCSt\ of rIlroIIIIclcIc nc~ti RS descrIhcd by SIARK#IAM ANI> cjSllTIlb

~~lbOrIl1CkJ2ILc~ WCrC prI2jXwcd If1 tlIL’ hbor.ltcJry in cry!+t~lillIIc fc)rnI fr0IlI Calf 1%~ncrc~s’, Or WCrC obt~inccl cttrnntcrcirtlly from Armour ant1 Cc3 , Worthrngton ( ltctn~cnl ( IS . .rntf iSutrItIona1 I31oche- mica1 C.rwl> , it* tn sm cwlicr InvcstqnlmP.

Ycnut I IlIr~nIIclcIc ncitl wa9 prcparctl a\ rcportctl* nntl ilcscr~l~ctl lU l>rcvIoIIulv

Ap~nrulrts I~lcctrophore~Is wa% .lcI.omplIrlIctl III phosphate buffer (165 5g of Nd4,1’0, H,O and 286 t I: of Nn,llI’O,~12H~0. rltr~olvctl In 10 i tlI~tIllctl wntur, ant1 1181 nrljurtcd to 6 49). usmg eehor (n) iln clcctrcq)lIorctIc appzrratIIs (Jr (b) the I,lCIS 3276 clcctroiIlrorc~Iu cquIpnrcnt

(a) I IIC cluctroplrorctic nppnrntus consist of il pnIr of I 5” n 4hfcl of \Vliatmnn ftltcr paper No t wncl hnntiwIclIecl h

squzirc yln~ plntcs. hctwccn wliich r7”

by CIIttIng oIIt il 2+ -5qlIilrO filter paper was prepared

“-square from each corner, SO that ia fiap was proviclccl on cnctr siclc of the glm~s plate* that wcrc piaccd In the buffer rcscrvmr coIItuInIng pli\tInIIIu clcctrotluv After the paper Wi\9 ccntcrctl on the platu. polyctlrvlcIic cover slrccts wcrc pl.rcctl on tlrc flap su th.lt tlIoy cxtcndcd nhout rg mm um the paper, and then the top plirtc WAS ndtlctl .rnci clnmpetl Spccrnl care was taken to pruvcnt Inflow and ntr hubbies

(bl in tlrc 1,ICIS 3276 cicctrophorcrIq cciIIIpmcnt I 70 Y 4 to mm \Vhatmnn fIltcr paper No 3 MM w.I)r II4ecl A I50 mm-aqunrc wns LII~ out from tlIc center 0f tiw paper, nncl n 8 52 mm-squsrc of WlIntm.rn fIltcr p,&pcr No 3 XlM wa\ piaccd over the cut atitrnrc

ElrclroplIorcstt In all cupcrInrcnts, tonrc cqtIIlIbrntIon \viIS accompitrhctt In 24 hours When IIeInl: lhc two ~I,Iw i>liltIY clcctrophorc~ts .lppnratrIs (n), 3 potcntIn1 of 300 V. 0 5 mA current, was first cqq~licd III cmc tiIrcctIon for 2.) hours at room tcmpcrnturc (approxImntc1y IHY) 1 hc current w:I\ stopped, anti under exactly the Yilmc c0nclItIanu. il potentrat of 300 V, 0 -5 mA crrrrcnt, was ~Lf~pllcti ilt riglIt nnyiw to the first current flow for ilIl .~ritlItIonni 3276 cicctrq~iiorc~Iw cquipmcnt. nftcr Ionic cquIiIbmtIon

24 hourr \Vhcn ustnp the LIil3 , ,t pc~tentini of 300 V, 0 5 nit\ current W&W

nppl~crl for 24 IIoIIrs, ‘I’hc current wit9 ~toppcrl, the I 5~ mm-\clIIare af Whattnan ftltcr paper No 3 &l&I wnh very cnrcftrlly turnccl 00 clcgrecs. anct a pIItCntI;tl nf 300 1’. 0 5 niA current was apphcd for nn i\tltlItlOn~l 2.1 hour%

GIrty8RIIc ncfturly 1:rom clIIplIcntc untreated clcctrogrnms triangular wctlgcs wcrc cut out of cacti of the four protein-contnimng scctrons I3y clIppIng the broad SIC~C (tcstcd for ;&+cncc of protcIns) of the paper In rlI?rtIllcd wntcr. the mntcrI,tl w,ts concentrated at the potnt of the wcdgc by captllary ,IctlnYl nntt bItcccY~Ivc cvaporiltlon hftcr conccIitmtIot~. the poIntct1 tip of cnch wcdgc WELS cut and placctl In iI \niall test tIItH: (ticsIpnccl for Inicronnnlysis) to wh~clr wa9 adtlctl 100 flrnl of 0 2M pho~phntt huffcr, pfI 7 2. contntn~ng 2 mg nf yctlat rIi8rmIIclcIc nc~tf, and IncIIhntctl at 37*C for 48 houra Ten PInI of chloroform wcrc uscfl ~3 n hnctcrrostattc agent

En!ymIc JctIvIty on synthctIc mcthn W(IR foilowcci In ii cnplllary tube. In wtrIch IOO ~1 of I?;, Jolutlnn of a cychc .lnlIytirIdc In 0.1111 ilcctiltc buffer (PH 6 $) was mIxcd wrth IO ,umi of protcln fraetzon 1. II, III, or IV (obtatnud by dIssolvIng the conccntrnted nrntcrtni at the potntcd tip of each wctigc as dcscrIbed In cnrlrcr scctton), ttus was Incubated at 37% for 3 hours, after scahng both cncts of tire cnpIiiary tube One ~1 of chloroform wsw uscct to prcvcnt bacterIaI growth After IncubtrtIon, tiIc capillary tube was broken open and 2 jrmi wtrc appllcd to Whatmon fIltcr paper NO. r of a previously cquthbratcd clcctraphorcsIs apparatus Controls consIsted of cychc nuclcottdc,

Ra~evouxs p. 446

VOL. 17 (1957) RESOLUTIOX Ol- CRY5TALLISE RIBOKUCLEASE 441

cxposcd to similar cxpcrimental conditions in abscncc of cntymc fraction. anti the nuclcotide cxpectctl as end pn-xlucts Elcctrophorcsis of this section of in\ cstig.ition was lwrformcil In an elcc- trophorczws n}>parntus ns ttescrl&!ed by I<uSKRL AND ~~‘Isr.~luS 1’ Satliiim tctrnbomtc. c) 4iIf. pH 13. scparatcil the cyclic anlr? Oriclc nntl the nuclcotrilc A current of 10 ni:\ ot I 100 1’. applicil for one hour. rcsultctl in il clear scparrrtton of the two jirorrp~ Scparntwn of ttrc 2 ‘-isomer from tire corres- poncltng 3’-lsomcr of each of the mononuclcotctlc N n- rcnllzctl by tlrc contlltlons dcscrlbcctl earlwrl*

Paler chromafogrnphv

< hrornatogrilms of the dlgcsts of ribonoclcic a00 X\ itli cncli rilwnuclcasc fractions (1. 1 I, 1 I I. ntlcl I \‘) wcrc ilevclopcd by tlic tlcsccndinjq tcctiniquc in t\\ 0 climcnsiocrs -1 Iic solvent ssstcms found to bcof vduewcrc solvent I ,anrmonin-l,iiftcrecl-?.col,iit\ r~cRciila4clcqcr~~~il by~~r\~~S~NlKefn~ 13, and solvent 2. rsopropyl alcohol-acetic mid-\vatW as ckscrlbcd 1,~ ;\I0NTRRulL AYI) ~OUI.hNGRR14 Per-

mnncnt rccortb of the chromatogmmq wcrc’ obtaincc\ by the tcchntquc of ;\IARKIIAM hsv SHITHIb

After locating tlic mononuclcoticlc nrezw. tlic*\ \\wc cut from the clironintogr~ms Conrldcte clution wiw ol)tninetl by pcrnritting t ml of tl~stillccl wntcr tr) drain o\*cr tlw filto pal17w into i\ grailiiatccl centrifuge tobc Each clunrrt \\n9 ntlJustct1 ftnnlly 18) ;I \ itI of 1 5 ml by tlrc tlw of n dtlutc solutlorl of I+CI in such il proportion tllat tllc final conc~ntr;~t~ou l)f IfC I was 0 01 S ‘l%c* ultrnviolct abqorp- tton nlwctrunr for cnch mononuclcot~tlr was clctcrnllnctl 1)~ tltc tlsc of n 13cckmntr >I~wlcl l)CI slwc- trophotomcter ant1 I ml rnpacltvcclls 13l.lnks wcrc rlu.tntn c$f tclcnttcnl filter papc’s cut flcnn hlnnk c1ironi;\togr~nr~

I< I.. S c’ LTS

‘I he rcc;olution of cq*stallinc rlbonuclc;\5c Into four cnayniica11~ xtlvc fractions Is prescntcd III 1;i.g 1 I~lcctrol~lw~ ew 111 the first clin~etwion l~roduc~d no rcsolut ion , IWase mo\xxI as a homogeneous lxotcin ‘I‘llc sccontl-tlimcnsioi1 clcctrophorcsis rcsol\ - cd IWaasc into four crw~tnicnlly actiVc lx-otcins

Crystalline ribonuclcascs ~aml~lcs, lx-elxwccl in the laboratory from calf pancreas, or obtained commercially from Armour md Co., \Yorthn@on Chemical (to , and Nu- tritional I3~oclwtnicnl (h-p , whcm c~\posctl to two-dinrcnsional 1xq-w clcctrol~horcsis were resolved into the cnz?,mically xtivc fraction5 dcscril~ctl in Table I Only the sample obtainccl from Xutritlonal 13ioclwmical Carl, was resolved into three compo- nents, all the other three samyles wcrc resolved into four components

Fig I Resolution of crystalltnc rlbonuclc.asc by two-dlmcnslonal paper clcctrophorcq~s (“detection reagent for protein-3”).

Refe*cnces p 446

442 A. A. HAKIM VOL. I7 (1957)

RJJJONUCLEASE ACTIVITY 01’ TJJR DJPJ’EHENT ACTJVJC J’RACTJONS Rl?SOJ.VJZD DY TWO-DJ.4lJ~NSJONAL

J’AJ’J:R J~LJXTROJ*HOHCSJS

“ArmolJr” RJlJon~tclcasc ‘*Worthington” RJl1onuclc~7sc “NutrJtJonal UJochcmJcnls”

Rtbonuclcasc *‘Ldxxotory” RJlxxJciclcdsc

JO0 48 1’ 05 25 98 100 4’1 43 3 z 97

100 50 44 0 95 100 49 4’ ; 1 97

_-___ -_ .-

Quantitative and quahtatlvc dlffercnccs are shown in the cnaymlc activity of the components of each sample.

Two-dimensional chromatographrc analysis of the hydrolytic products resulting from cnzymc actron of the four cornponcnts of RNase on yeast rlbonuclcx acid, using the solvent system anlmonla-buffcrcd-svobutyrlc aclcl, and zsopropyl alcohol-acetlc acid-water (Table II), revealed some lntcrcstmg cluantltatlve dlffcrence m mononu- clcotides liberated Ribonucleasc IV hbcratccl more guanylic acid (lo.5 molcs/xoo mo- les) than RNasc III (2.5 molcs/roo moles), RNasc II (o 5 molc/roo moles), or RNase I (0.5 mole/xoo moles) Rlbonuclcnsc III liberated more adcnyhc acid (8.5 moles/roe m&s) than RNasc IV (-, 5 molcs/xoo moles), RNase I I (2 > molcs/;oo molcsj, or RNase I (0.5 moles/roe molts). Ribonuclcasc II hbcrated more cytldylic acrd (x8.5

TABLE II . ACllON Olr CRYSTALLINJ, I~1IJO.SUC,Ll?hSJS I’RACTJONS ON YI.AST RJJ3O.NUCLRlC ACJD

Fror1tont

~lononuclduier ltbrrcllrd _ .--. ..__ _ .._._. _ ___ ,._.. -__-_- -_._ __ .__-.. -___ WldyltC cylIdj&- - adrnyltc

actd odd act&f C@zzP mob per 100 m&l

--

1: J7 b J7 4 05 05 170 J8 5 05

III JS G ‘4 5 i5 25 IV JJ 5 Jo 5 4 5 Jo 5

moles/xoo moles) than RNase I (17 4 molcs/roo molts), IiNasc 111 (14.5 molcs/xoo moles) or RNase IV (x0.5 molcs!roo molts).

Paper elcctrophorctlc analysis of the hydlolytlc products producccl from cnzymc action of each of the four rlbonuclease components on the synthetic substrates, urldinc- z’,3’-phosphate, cytldmc-2’,3’-phosphate, adcnosme-2’,3’-phosphate, or guanosmc- 2’,3’-phosphate (Table III), mchcatcd that the major activity of RNase I was on un- dine-2’,3’-phosphate, RNasc II on cytidme-2’,3’-phosphate, RNasc III on adenosme- 2’,3’-phosphate, and RNasc IV on guanosine-2’,3’-phosphate.

RNase (Armour) solution was kept at o”C, and at period intervals, aliquots were assayed for enzyme activity and for ?wo-dimensional paper elcctrophorcsis. Enzymlc activity was almost constant, dlminished at most by approx. 2‘1: after GO days at 0°C. Paper clcctrophoresis revealed some quantltatlve difference in the nbonuclease components. RNase I and RNase I I showed progressive decrease in activity, from 48 Ralerences p 446

17 kls7) RESOLUTIOS OF CRYSTALLINE RIBOBUCLEASE 443

FIR 2 hctmn of the four ribonuclcn+c poncnts on urldmc-2’.3’-phosphate

conr- 1:

1’

. .;_.. ._ . ‘~g 3 Actloll of the four ribonuclcasc oncnts on guanosmc-2’,3’-phosphate

C om-

to 38, and from 41 to 37, rcspcct~vcly , RNasc III and RNase IV actlvitics progressively incrcascd from 6.5 to r I, and from 2.5 to 9, respectlvcly

Pig 4. Action of ribonuclcasc components I and IV on yeast tlbonuclcic acd Two-dlmcnsto chromatographlc analysis of the hvdrolytx. products rcsultmg from tha actlon of rlbonuclc components I and IV on yeast nbonuclelc acid.

Rc/srenccs p. 446

‘nal iw

444 A. A. IihKIhf VOL. 17 (1957)

ACTIOS OY CHYSTALLINL HII5OSUCLI:ASI: I’HACTIONS ON SYNTHETIC SUWXRATES

I GO cm=-3’ 80 UMP-3’ 0 - 0 -

II 78 carr-3’ 65 UMP-3’ - 0 111 45 CMP-3’(-2’) 40 UMP-3’(-2’) 0: AM P-3’ ‘5 GXlK3 s IV IO CMMP-3’(-2’) 15 CMI’-3’(-2’) 20 AMP-3 55 GMP-3’

- --

Av~k cnw-3'. UhW-3’. AMP-3 ant1 GSIP-3’ arc the 3’4crcvatIvc3 of cyt8tlylic. urdyhc, adcnyllc and guanylic mxd rcupcctlvcly

In the prcscnt mvcstigatrons, crystallme ribonuclease has been resolved mto four malor components by two-dimensional paper electrophoresis. The four components have been shown to possess difference m cnzymic specificity.

Our analytical data mcllcatcd that each of the ribonuclcase components is mvolved m the hydrolysis of specific mternuclcotide lmkagcs; RNasc I and RNasc II hbcratccl more uridyhc acid and cytidyhc acid, and dcmonstratcd a greater cnzymic activity on uridine-z’,3’-phosphate and cytidme-z’,3’-phosplratc than either RNasc I I I or RNasc IV. Conseclucntly, RNase III and RNasc IV hbcratcd more adenyhc acid and guanyhc acrd from yeast ribonuclcic acid, and demonstrated a greater enzyrntc actrvity on adcnosinc-r!‘,3’-phosphate and guanosinc-2’,3’-phosphate than either RNasc I or RNase II.

These variations in enzymic activities of the four malor fractions, RNase I, II, III, and IV, provldc strong evidence for the suppositron that crystallme ribonuclease has different active fractions, each with its own specificity. Each specific fraction contri- butts to the hydrolysis of ribonucleic acid, and to the cleavage of specific mternuclco- tide linkages m the ribonucleic acrd molecule.

The mechanism of action of rlbonuclease is not a simple hydrolysis but an mtra- molecular transphosphorylation followed by hydrolysisi@. KUNITZ~ demonstrated that the rate of hydrolysis is slower than the rate of transphosphorylation. The resolution of RNase into four active proteins indicated that each of the protem fractions isolated had two active sites, a transphosphorylation and an hydrolytic site. The electropho- retie resolution of one active protem into four fractions, each revealing a certam de- gree of specificity, suggested that the “actrvc center” of brologically active proteins lies in some correct spatial configuration of polypeptide chains. Although this finding is m agreement with reports in the litcrature17a18, it demonstrated specifically that certain polypeptrdes m an active protein molecule may reveal certain specific activities that could differ from the whole protein molecule activity, and thus the activity of the protein molecule 1s the resultant of activities of its polypeptrde chains.

The degree of heterogeneity of the preparations studled depended upon the condi- tions employed in the isolatron and crystallizatron of the enzyme, while quantitative amounts and proportions of the four major fractions revealed the age of the enzyme Re~svsncss p. 446

VOL. 17 (1957) RESOLUTION OF CRYSTALLINE RIBONUCLEASE 445

(Table IV), and the degree of oxidation or reduction le. LEDOU@ has suggested that the chromatographic inhomogeneity of RNase might be attributed to different stages of oxldoreductlon of Its sulfur group21~~.

TABLE I\’ Kl’bRCT OF AGING OS WLBONUCLEASE ELRCTROPHORETIC HETEROGB.NflTY AND I’.NZYMIC

ACTIVITY’

Fresh RNase solutron h RXa.x solution 24 48 h RNasc solution gG h RNasc solution

192 h RNax solution 384 h RNase solution 768 h RNasa solution

1536 h Rh’asc solution

Note The relatlvc cnzymlc actlvrty of fresh, 24. 48, 96, 192, 384, 768 and 1536 h solutrons wcrc 100, 99 9. 99 8, 99 8. 99 8. 99 6, 99 o, and 98 o y;, of the orlgtnal actlvrty

l Crystallrnc “Armour” cfystallinc “Armour”

rrbonuclcase. rccrystalhzed twlcc bcforc use Two per cent solutron of rlbonuclccuc m a 25 ml flask The surface of the rtolutron was covered with

z mm of mlncral 011, thus protcctcd from atmospheric contamtnatron At period intervals ahquots for cntymc aquay, and for paper clectrophorcsls

Rlbonuclcaqo actlvlty was dctormlncd by folIowIng the optIcal dcnslty of mcthylcnc blue (o 20

g/ml) and rlbonuclcrc nerd (5 m&ml) at 5500 r\ a4 a function of tlmo. after the nddltlon of the different rrbonuclcase solution or fractlonLJ

SUM,MAR\-

1 Crystalhnc rlbonuclcasc samples obtamcd from dlffcrcnt commcrclal sources In oddltlon to one proparcd m the laboratory wcro rcsolvcd mto thclr components, RNascs I, II. III and IV, by a new two-dlmcnsronal clcctrophorctlc tcchmquc

2 RNasc I and RNasc II hberatcd more urldyhc .rcld and cytldyhc acid from yeast rlbonuclclc acid. and domonstratcd a greater cnzymlc nctlvlty on urldrnc-2’,3’-phosphate and cytldlnc-2’,3’- phosphotc, than either RNasc III or Rh’ase IV RNasc III and RNaso IV hborated more adcnyhc acid and guanyhc acid from yeast rlbonuclclc acid, and showed a grc,lter cnxymrc actlvlty on adcnosmc-z’,3’-phosphate and guanosmc-2’,3’-phosphate than either RSase I and RNnse 11

3 The dcgrcc of hctcrogenclty of the RNasc umplcs studlcd rcvcnlcd the age of the prcparatlon 4 It 1s thus demonstrated that ccrtatn of the actlvltlcs of ‘%rystalhnc rtibonuclcasc” rcsldc tn four

dlfforcnt protcm entitles, and some activrty toward purmc nuclcotldo cstcrs cxlstctl In two of the four protein entltlcs

I. Des dchontlllons do rlbonucldase crtistalhsdc provcnant solt du commcrcc, sort dc prdparatrons dc laboratolra, sont d6compos6s par uno nouvcllc mdthodc d’6lcctrophor&sc sur poprcr, A dcux do- mansions. cn rlbonucl&so I, II, III et IV

2 Lee rlbonucl&ses I at II provoqucnt unc augmcntatron dc la qunntitb d’ac~dc uridyltquc ct d’acrdc cytldyhque lib&r& A partir dc I’ac~dc rlbonucldlquc dc lcvuro ct montrcnt unc plus grondc actlvltd cnzymattquc sur l’urldmc-z’,3’-phosphate ot sur lc cytldmc-2’,3’-phosphate quc Its rlbo- nucldases 111 et IV Lcs rtibonucldases III ct IV hbercnt davantagc d’acldc addnyhquc ct d’acldc guanyliquc A partlr dc I’acldc rlbonucldlquc dc lcvurc ct montrcnt unc plus grandc actlvltd cnny- matrquc sur I’adtnosmc-2’,3’-phosphato ct qur lc guanoslnc-2’,3’-phosphate que Its rlbonucl6anas Iet II

3. L’hBtbrogCnbrt6 da la ribonuclbase btud& rdvL\lc 1’5gc de la prbparatlon. 4. 11 a 6t6 d6montrd quc ccrtamcs actlvlt6s de la rlbonuclbasc crlstalhsCc rddlsent dans 4 pro-

t6mes d:ff&entcs, ct que son action A 1’6gard dcs cstcrs dcs purmcs nuclCotldcs cst duo A 2 seulemcnt de ccs prot&nus.

Rsfsrencss Q. 446

446 A. A. HAKIM VOL. 17 (1957)

ZUSAMMEN FASSU NC

r Vcrschlcdcnc 1~;1ntlclupr.lparatc SOWIC such lrn Laboratoraum hcrgestclltc Probcn von kris- lnlhslcrtcr Rlbonuklcasc wurdcn mlt Hllfc* clncr ncucn zwcldlmenslonalcn Elcktrophorcsomcthode 111 ~hrc Bcstnndtcllc, Rlbonuklcasc I, II, III untl IV, gcspalton.

2 lilbonuklcasc I und II sctrcn aus Hcfc-Rlbonuklclnshuro mehr Urldyl- untl Cytidylsaurc In ITrclhclt untl /crgcn clnc grossclc cnzymotlschc Akttvltat gegenubcr Urltlln-z’.3’-phosphat und C\,tltlln-2’,3’-I)~~o~l~~~at als Rlbontrklcasc II 1 untl IV Dagcgen setzen dlcsc bcitlcn Ixtztcren mchr AC\;lcnvl- ontl GuanylyrLurc sub HcIcr,bonuklclnsaurc In Frclholt und zclgen cinc grdsscrc cnzyma- tlschc Aktlvltkt gcgcnubcr Adcnosln-2’,3’-phosphat untl Guanostn-2’,3’-phosphat als CIIC bctlclcn Erqtcren

3 DIG Unctnhothcllkclt tlcr untcrsuchtcn Rlbonuklcascpr&paratc war cln .Maw fbr dcrcn Alter 4 Es wurJc dargclcgt. thes clnlgc hktlvlthtcn tlcr ,, krlstallnuertcn Rlbonuklcasc” /.u v~cr vcr-

schlctlcncn Protclncn gch0rcn und class ZWCI von thcscn vlcr Protclncn one gcwlsqc Aktlvltot gcgcn- ubcr I’urlnnllklc<ltltlcstcrrl bcslt/cn

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Rccclvcd Aprrl zbth, 1957