Fz>ort of' LE. kvery (assisted by Dr. IflcCarty)


      S tucis>s Or1 ~X~~UEOCOCCUS .  The results of earlier vtior*k on the
           --.u-

chemical 11:.tur0 of the substance inducing trans.format.ion of pneumococcal

types ;yil-c i;l*idfl-r summcirizoci in the lest report to the 2o;lrd.  This year


c p';per i:C:rlitlg in n0r.e tict::il wittl the chemical pr;/,;,;i't.i*2s and biological
                                            ,
~(J~Ci~`iCituy fir` th:: aciivc princi;lle arcs publi;;hd:c in the FcbruLry issue of

the Journs of Xx~elirni-nt;,l m%icine.  Further* uoric is no\; in progress on
                          ------

cert:-in 2k-.I;ticts of thcl problem, the cl.uci.d::tion of crhich, it is hoped, may

proviae ;~cclition~i cvidelici: in support, of Ihe belief th:-t il. nucleic acid


of the r.lt:soxyy.j bosc tyy Jt' is the fundamentsl unit of the t,rttnsforming agent.

In order to establish tilt vrlliditJV of this concept zno to Lcquire a fuiler

knor,l:sdg:2 of the bioc.;ierriicai i':;ct.rjrs ir.volvtid in the ~~honomt;non o$ trans-

formation in vitro, .the L'oiloy,ing problow ttre under: in-~esiigxtion at the

present time:  (1) th; isolat.ion clnd purificstion r^rcJm :.ni%l tissues of

the anzymo cap;ibla of ticpolymerizing da~ox~-riuonucleic z:citi; (2) the re-

versible inSactiv<Ation of the transf'orm.ing principle by known chemical

substances.


        1.  Desoxyribonuclaodepolymersse. in the ptipar referred to

above it wtis pointed out t1xit among various crude preparations of enzymes

tested only those thtt proved cap:&bILe of depolymerizing Guthantic samples

of desoxy-ribonuc.l.eic ucid were found to destroy the biological activity of

the triaiforming subs taco.  This KGS true irrespective of the presence of

other enzymes such LS prote;:ses, phosphatases, or ssterases ;.nd regardless
of whether the prt!perz.ti&s were obt;linod from pncuiococcal cells, from the

blood se-rum LX urg?ns of dir'ferent munmaiizn species. The fact theit enzyme

preparutione which detroiymerize desoxyribonucleic acid uf animal origin

also inztctivrAte the trasfiwming subutr;nce and the :I.dditional fact that


the en;;ymes invo!.vec.l in tio*,tl of those re;;ctiono sre inactivated Ltt the

shine tti,;ipor::trui.t. i;r~d :;ro inhibil.ea by socii:x~ fluoride zre I;recum~tive

evidence t&t tke trrinsi'c;rsi.ng principle itself is ;I nucleic hcid of t'he

cestly*irj &S,z
3     .u'      type.  ',or.sequently, it seemed of prime iirii)OrtUlC~ to isolcte

this tlnzyme in i,ure f'or.m, i'ree from other ens;~~es, in order to pl*Ovt: tkt

the loss of tXWisfOrIIling i;C Livity is kl:tucl?Liy due tfJ b single mzyme

ticking s,kxil~icsi.Ly upon tile b;ct,cl i;ll nut Lc:zte which is btiiiev!$d to be


iaenticiil v:iLh ;he trt,ri~~'or:ninL; substkrice.  if unaer these conuitions

d2pOlJiilc~iZi; Lion I*t;sUitcd in icJsb of bi.olOgic2.l r.ctiviky, it wuld. provl?

beyond retisonk ~11~ coubt !.h t, the nucleic iicia kina the trxx5forming agent

are one Fncl LkiC? SLilrO sutxt;.~nce.

      Prep)rrriition:j o?' 6,::; oxyr..i.bonucic-ouep~~iyi~l~r:;s~ extrtc Tad from


                                 . . .
commercis k i,i:nCri:;'iin cnu ~JiL'J.1 ICC [jJe Pr;lc i;j onai. ~~r~eci~~i.t~i,tion with
iQ$X14 hxvc been found to p,~:~s*s:I high tictivity in coricantrhtions cs low

as i $gm./cc.  The :.ctivitJ of' the enzyme is moasul.ed by its kpiicity to

reduce the viscosity of hig`tli;y polymcrizea dcuoxyribonucleic :.cid isolated

from calf tbymus.  bimiiar:Ly, minuta :. mount:; of enzyme protein httve been

shown to bring about rapid ;:nd colrtplote inactiv&tion of the Lrunsforming

substance.  lir. McCarty OILS aeveioped quantit&tive methods for the measure-

ment of enzyme activity.

      hiugnesiuni and mkngancse IEtve been found to activate the depoly-


mercise.  The fact thut ivlg SWVU:, us nctivator is in xcora with the finding

that fiuoriae inhibits the :&Con of this anzymc.  In the case of other

enzymes which elre activkteti by Mg:, the inhibition .by fluorick is attributed

to a combinrrttion of m&gnesium, fiuorj.dc tinti phos;+hute \;hi.ch removes the

magnesium ions from p,tirtic.ip:ALion in the enzyme action.  Bw2;tuse of the

fkct that citrate is Known to form h soi.ubi2 ccjrrqJ?lcjx with caicium, it


                                                  ijj

seemd jjo5sible th2t a siiuikr rextion ini.i:ht occur with mqnesium und

thclt cl;nsequ+rltijr citrate might serve to inhibit the action of' the enzyme.

This W&S fauna to 'w the case, rind mhrkad inhibition of de;.,olymeri!se

aCt,ivit..;r csn be effected in ihe presence of citmte.  Limi.Lcjrly, the in-

xtivktin.- effect
          e      Of UCi,Oljri!lC~L.Z.Jf2! on tfit3 t~;r!lS!`~~i?lIl~ ~libSt;..I~C!~ is in-

hi`oiteci by cil;r;lie.

       In tht! pl'~Sent htLf?IliPtS TV pui*if;r tht: aepolymcrase it hss not

hs yzt b$zen possible to free the peyxxtiun compLete.Ly of all. trxes of

b. trypsin -Like enzyme.  'T'na proteolytic !.ction of the kLttzr results in

c0nsiawtitl.c: loss irk activity of thi: depolpnerhse durin,? the preparative


proceauros.  ;;ietliObS for OVerCui;lin& thi Y ciiff'ir;u.lty hnd selective mei\ns

for septrEiting the depo.ip~~*asa in pxrc fort;; tire uncicr investigation.

hli.hout;h try,jti<: enzy:::.:s try r,xb<:ilt., tht-r2 is rLmpl.c c-Jiuence t.hilt they


hre not rl;sp,onsihlu for inLctivL.tion of she tr~,nsfor*ning principle, since

it h&s been shovm that n&.thcr cryL:Xl line trypsin nor ck,ymotrypsin hiis

uny ciemonstrh blr! effect on trt~n~for;iling sctivity. Lioreov~r, these prote-


ascs 81% not inhibittiLl by fiUGl'idi: or by citrrlx.

         2.  heversible inxtiv&Gon of the trtinsforming subst:ince. In

the course of isolation tuna +.ri.ficktion of the tr&nsforming Ligent it WLS

found that pneumococcsl extracts treated with t;scarbic acid completeiy

lost their transforming activity.  A more detailed study of the nature of

this inwtivution is now being carriea out using highly pwified yrepara-

tions of the triLnsforrning principle.  k solution containing 3 mg./cc. of

fictive substunce, of which ti.02 pgm. wffices to inauce trhneform+:tion, is

COIilpiet61y inactivated after* contxt for 5 minutes with ascorbic acid in

0.01 M conceutrltion.  'I`rr,crs of copper which ure known to c;tzlyze the

auto-oxiustiori of iLscorbic ;tciu h>.ve iikwise be2n fwnd to r=;ltslyze its

action on tAe trtinsforming i-gent.  A coucC;ntzLt ion of 0.301 ,ti ::scorbic


                                               i-36

acic, which by itself brings a'sout oniy partial inactivation, causes

complete loss of jciivity in the presence of minute traces of co??er.

      Hopkins and rv!organ have sho;vn that recluceo glutathione completely

prevents the oxidation of ascorbic acid even v;hen the lutter is cataljrzod

by cogG;er ions.  ks litfle as '3.iUj. Li concontr::tion of zlut&thione has

been founu to ,zotect the transforming principle Prom inactiv&tion in the

presence of O.i)l ivl ascorbic aciu. Thus, it tliJ&)i-'e::rS th:Lt the oxidation of

the ascoi.bic ;cin is i neceb5ary step in the inactivation of the trans-

forming ai:*nt .  iriore signit'icclnt is the fact that transforming substance

which has &:i:n corni,ieteljr inacLi;lated by ascorbic ucic can be quuntita-

tiveiy retctivsteo bJ subsequent tretrtment viith glutathione.  Thus, the

rebction is h rcvsrsibie one Lppc+I'Cntl.y ue~end2nt c;n the oxiarztion tind

reuuction of' bioiogichily irapoi'ttirlt groups in the nucieic hciil molecule.

ii vtlrieCi of' otntir oxidatits, such as ferricyanide, cytochrome C,

flavine phosphate, and a-tocopherol iA~osph;lte,  testeti under similar condi-

tions, do not ;ffect the activity of the t~:.Lnsf'ormir!~C; aubst;nce.  On the

other htind, i;jo- and g~ucosscorbic Lcid, which possess little or no

vittimin C activity, are just as effective in bringing about the inactiva-

tion of the tr&nsformi.ng substance as is ascorbic acid itself. The in-

activation induced by these chemicai analogues c;an be prevented when the

reaction is carried out in the presence of glutzthione.  Among other

substances tested, catechol has been found to behave like ascorbic acid

in producing reversible in&ctivation of the transforming substance. It

is noteworthy t&t catechol, ascorbic ticid and its aerivhsives all possess

in common ciienol groups, th&t their action is ccitalyzed by copper ions,

end thst their effects can be reversed bJr giuttthione.  The loss of ac-

tivity inouccci by these tgt-nts czn be restored b;r 0~: :&ction of other

sulflvoryl coi~~pounca tuck! ils cysteina i~nd thioglycolic acid, although


b