Itcccntly reported cspcrinicnts li:t\rc r.ix!c it
llc~~~~ary to nssunic t1i:rt Llic iiiducctl sj.iitlicsis
(,I cnaynics in microorgnnisnis is coupicd ni::n-
,]!rtorily to tlic utilization of frci: arnico :icitls.
1:ivest.igations (Ralvorson :md Spicgclni:ui, 'ICE)
*xit'n ycsst havc shown thnt nnalogucs of :imino
:&!s Jvhich prevent incorporation from tile frcc
finlino acid pool into protcin :LIJO suppxss tile
illtiucctl forinntion of a-glucositl:w. Ikthcr-
more, 3 coniplctc p:udlclisin bctwcm a-glucosi-
(!~.SC synthcsizing cnpncity and tlic av:?ilablc
intcrn:il supply of free nmino acids WIS tlcrnon-
*tr:tted by nicans of dcplction ant1 rcplenishmcct
cycles (IInivorson 2nd Spicgclninn, i053a).
Finally, cmploying the-samc system,  it was
possible to esliibit (Halvorson and Spiegclman,
1053~) n net incrcnscd utilization of the ictcrnnl
frcc amino acids 3s a consequcrice of the induc-
tion of cnzymc synthesis in cclls suspencicci in a
Iiicrogcn-free medium. The generality of the
fini1i:Igs with ninino xid mdogilcs was c:itenilcd
ii;. tlic independently pcrformcd cxperinients of
I.cc uiid Williams (1952). These authors dcmon-
stdctl that the administration of ethionine to the
int:ict rat preventccl the induccd formation of
tryptophan pcrositlase.

Monod el al. (1952) studied ihc fornintion of
j%g:ikctosidnsc in a scrics of aniino acid RIISO-
tmpiiic mutants of Escltcriciia coli. In no case
WLS enzyme synthesis obscrvccl in the absence of
the particular amino acid rcquirad. Iiickcnberg
rl al. (1953) rcport esperimeiits aloilg similar
lints in which @galactosidase formation was

'This research was aided by a grant from thc
Siitiond Cancer Institute of t.hc Natioiinl Insti-
tiites of I1calt.h and by a contract between the
Office of Xnval Research, Dcpnrtment of the
Sitv-v, find the Univcrsity of Illinois, NR 135-136.
*A sumninry of tlic prcsctt rcsults was prc-
:c.ri:cd at the 1953 inccting of the Society of
.\m&nn Bacteriologists (Bacteriol. Proc., 1053,
1'. 2).

* Prcscnt address: Enzyme,Institutc, Madison,
Kiuconsin.

-

fol!owd in mutant strains of E. coli, strain
IC-12. Analogous rcsults ah wcrc o1)tainctI in thc
intiuction of a myoinositol degradation system in
Acrobaclcr acrogencs (Ushiba and Magasanik,
1952).
Thc dntn cited would appcar to eliminate any
nicch:inism of enzyme synthesis which involves
an amino acid indcpcndcnt irmsfoi nintion of 3
prccsistent coniplcx prccuisor into active
cnzirnc. Tiicy do not, lion ever, ninkc unten:iblc,
or dist;nguish bctwcn, t?ic following two pos-
sibilities for enzyme synthesis:

(a) I'recursor and iicc amino acids --* Active

enzymes.
(b) Frcc aniino acids -t Active enzjme.
It is thc purposc of the present paper to ofier

datn rclcvnnt to this issuc. The most obvious
experimental approach aimcd at a decision be-
tween the abovtf two nltci natives  oirltl nppcar to
112 the use of isotopic I:ibels. T~IUS, the induction
of cnzyriic synthis in uniformly lahc!ctl cclls
suspidcd in unlnbelcd medium should provide
the necessary data.

It is cvidcnt from the type of esperirncnt con-
tcniplatctl that the presciice of 3 laigc internal
frce amino acid pool, such 3s exists in thc pe,zsts
and many gram positive bacterin, ~vould intro-
duco a complication which could \veil make tlie
data uninterpretable. To avoid this complication,
attendon was turned to enzyme forniation in
gram negative microorganisms which havc bccn
shown (Taylor, 1947) to lack dctectablc frce
anlino pools. The system selected for study was
the 8-galactosidase of E. coli. The choice of this
particular enzynie was dictntcd by a variety of
reasons. Lederbcrg (1950) dcvised 3 convenient
and accurate assay of its activity by employing
the sj-rhhebic chromoganic substratc o-nitro-
phenyl-8-~-galactositle. In addition, the intensive
invcstigations to which this system in E. coli h:ls
becn subjected in reccnt ycars have elucidated
not only purely enzymatic propcrtics (Colin and
Monod, 1951; Iiuby and krdy, 1953; Lester and

419

IIoii~i~r, ICJ~~; I,c>~cI., ICJ.??) IHIL al:o IISVC ])to-

vitlctl t1ct:iiictl iiifw:n:itirJn on tho gcmtic
(Lcrlcrbcrg, I!jc??), iniriiiino!cb::ic:J (Colin :ind
Toi.rinni, 1952, 19.%$), :inti LIi!: spccilicity as1icct.s
of tlic inducti\,c process , (Monoti c! al., 1951 ;
Koppcl c: al., 11353).
The cspcrimcnts to bc dcsciibzcl i:i tlic prcieiit
paper Cl11i)lCJ~cd Ci4 3s tlic iscito;)ic i:ibc:. The
rcsults obt:ii;ictl 1cad to tlic c~~~iclusi~~ii iiint tlicrc
is litt!c or no prcforinctl coniplcs Imcursor wliich
is convcrtctl into nctivc cnzynic. ~\ii:ilogoiis
lintlings :id similar conclusions wrc :rrrivctl :it
siniultancouslg by Cohcn :iid IIugncs (for :i
prc1imiii:try account, scc il.Ionod and Cohn,
1953). Tlicse authors employed SJ5 as tiic isotopic
label and predominantiy immunoiogical pro-
cedures, for the isohtion of the @-gdactosidase.

BIATERIALS Ah-D XETXODS

Orgnuism uscd mrl conditions 01 groxtli.. Strh
h?L of E. coli w:is uscd. It  is 0I~L:iiiicil through
tlie courtesy of Dr. S. 13. Luria. Stot!i cultures on
syntlictic nictliuin scr\~tl as tlic sourcc nxiterial
for the prcpnration of cspcrimcnts! suspcnsions.
Unless othcrwisc spccificd, 3 modification (rc-
ductiori of the CaClz by onc-half) of tlic synthetic
medium (&I-.%) dcscribcd by Monod el al.
(1951) was used. A suitable carbon source was
ndtlcd to thc above incdium :rnd thc culturcs in-
cubiitcci zt 37 C.wit1i vigorous nerjtion.

. Circmical and rarliouctiac rccigints. 0rtlio-Xit.ro-

according to the nictliod rlcscrilccl by Scitlmin
and Link (1950). AIclibiose \vas prqxircd from
rarjiiiosc according to tlic proccc!urc of I-Iudson
arid Hardin;: (1915). Unifornily Ci4 1:ibclcd su-
crose \vas prcparcd from CL400? by tlic iisc of
leaves of Ctinna indicus (Gibbs et nl., 1952).
The G14-sucrosc was convcrtcd to lnctstc by
carrying out n fcrnicntntion with Strcptococcus
faecalis. L-Lactic ncid (Pfmstichl) was cniploycd
ns a carrier in the prcpuation 2nd purification

All the water used in thc present investigations
was double distilled from a glass still.
Anal$ical proccdures. Lactstc was analyzcd by
the method of Barker and Sunimerson (1911)
and nitrogen by the ncsslcrization proccdurc of
J,:iiini el al. (1950). In the I:ittcr, color wis
mc:rsurcd at 500 nip in a Bcclmiau spcctrophotoni-
etcr using 0.5 nil cclls. For prcliiniiinry survey
experiments, the micro-Fohn iiictliod (Lowry

I'helIS'l-~-u-~:tI:tctosidc (OXPG) ~x;::s s~Iitlic.jizcd

(Wood el UZ., 1945) of the hbclcd lactate.

cl G!., 15.51) for the cictcriiiiri:ition of protein .'.-
uwd.

1 l: tdio:ict ivity I1ic:isurcnicii ti \vI.:~c n:::(:-< .: :. ,
3 "Xwlcu-" Q-ps flo~\. cocntcr \viiicil I)(,:~, .
:L Lxkground count in the r`ci$l~odicm~J of 1:; :.
20 cocnts pcr min. Tire spccific rsc!io:lci;t-i, .
and, hcnce, tlic :mounts rcciuirctl for wl~;L+.
mcnt U-CTC such that corrections for sclf :I(!
tiorl ~vcrc only iieccssnry occoaion:iily. C(,iii,::: ~

\vas coritiriuctl until I.)ct\\.c~ii 1,500 :~ntl 2.c.:
COIIII~S h:d :icc:irrnul:rtcd. 111 certain VK;, 1. ,

C;ISC:,, longcr counting tinics wecc uscti tc, i:.
crc:tsc tlic :rccumcy.

To obtain an accurate cstimnte of the siJ(:;::
rndionctivity of the protein in the cluntcs !rt-.
t.hc st:trch column, it was necessary to rci:,i,:..
adventitious nonprotein cnr`bcn. Purificatlo:1 ci
tlie protcin was achievcd by precipitation !%,::I
trichloracetic acid and sdxcqucnt cstrsr:::
with hot trichloracetic acicl folkJl\.Cd hy ibii!,. :
cstuctions with ctlicr-rncthxol niisturef. ?,
typic31 purification of this naiurc nxiy I)? ,:..
tailcd as fdlows: To two ml of an clunte fi~:~~
stud1 column is addcd 0.3 nil of a 50 pcr cc;~
trichloracetic ncid. The resulting prccipita.;~ i?
spun down, and 5 nil of 5 per cent trichlorxe::;
acid addcd, allo~ved to estract for 25 hr at IO.:,:..
tcmpcrnturc, and then brought to 90 C and
for 10 min. Thc precipitates nre centriiugcd :kc:,
arid cstrxctcd twice with 5 ml of a 1 : 1 niethn:i..:
ethcr misture. The residue is collectcd Iiy ci:..
1;rifug:ition :ind dissolved in one nii oi
N:LOI-I -ivhich subscqucntly is ncutraiizctl \\~:i
HCI.

6nzynic was nss:iycd by a n-iodification ol :!.c
proccdurc suggest& by Lcderbcrg (1950). It wL,
found tliat, whcrcas frcshly rccryst.allizc-l
o-nit.rophcnyl-P-D-~~lactoside wk hydrolyze6 i::
tlie presence of enzyme linearly with time, dix.
picies from linearity wcrc obiainccl nitii oldc:
prcpsrations. It was cst:iblishcd cmpiric:illy tL:
the incorporation of yeast estract in the :IS-:;:;
preparation counteracted this cflect. TO T.Y~
the necessity of continually recrystallizing t1:c
reagent, ycast estract wxs inGudcd. The I:\tt<i
~3.s prepared from pressed baker's yeast (F1eisc.t-
mann's) in the following manner: 54 g of pt
are sus+cnclcd in 20 1111 of &O, autoclnvcd at 15
lb :Lnd 120 C for 0.5 hr. The rcsultant cstract is
ccntrifugcd, the supcrnatc llrought to a Lo;,
trcatcd with decolorizing carbon, and filtcrki
The filtrate is stored in the decp freeze.

`r~,,, wzpc rcnctior. \v:is Ti:[: :!t .!3 C 111 tlrc
+,qice of 0.001 IC o-~iitr~,j~!~,~ll~~.,~-i~-~~~l:~~~~~~~~~~
:!;-+j~vt>(~ in :L niistui;c of s;i;12i'()l ::wj s:~~I!I'o~
:,, yicI,l  diit.iol1 ;Lt pIi 7.5, 0.1 ;; t:;i:l\ rcspcct
L,, S:I, :iil(l contniniiig 0.0.1 in: G: yc;i..t c:;Lr:iet pcr
II:/. Thc tlcvclopzicmt of color \*::is idowcd hy
.14ip:,:1s of n T<lctt-Sumiiici;:on cnloririietcr using
filter no. 4%. One unit of cn;:gmc activity is
~~;)r~sc1itcd by t.lic hydrolysis of ow rqu mole of
`U.llit~,i[JIicri~i-~3-~-~nI:~rtositlc per r.:inutc unticr
::,c :,\~)vc conditions. Spccific xctivity is rcportctl

111 ~lriits pcr pg of nitrogcn.

I'n-(Jn ration oj pirrijicd u xt iscr II n~. Thc nic thotls
crliploj~tl for thc prep:mtion and purification
,,f nnti-p-~nlactosidnsc antiscrum \l`\`eic dcrivcd
jrorli the invcstigntions of Colin and Torrimi
(1952). P-Galactosidnse purifictl as dcscribcd
~,C]OIV \\-as used as the antigcn. Approsiniatcly
0.9 mg of purificd nntigcn \vas injcctccl into cach
r.ii)bit with mineral oil as nn adjuvnIit (Freuncl
ll~itl Bonnnto, 1944). Sera wcw co!lcctcd, pooled,
2nd t.hc y:-globuliu fraction isohiccl by cthnnol
fractionation (Xichol and Dcutsch, 19!S). Use
oi such prcparations avoids tlx nonspccific
prccipitntion which. oftcn occurs on * adding
hctcrinl cstracts to wholc scrum. Thc con-
ditions dcscribcd by Colin (1932) and Colin and
Torriani (1353) for obtaining specific prccipita-
tions of p-galactosidase %it11 antiserum wrc
follo\vcd.
1'uriJicrtLion of fl-gahAos.idnsc. `fhc purpose of
tlic csperimcnts to bc dcscribctl dcnisndcd a
proccdure for the isolntion of pure enzpc from
small nniounts of matclinl 1v1iic.h woiiltl bc
enicient in thc yiclcl of final product md lcad to
n iiiininial amount of inactivation: Thc latter n`as
necessary to avoid tlic complications in spccirtc
activity determinations diicii woulil derivc from
tlic presence of enzyme protcin not idcntifiable in
ternis of enzyme activity. It was cvidcnt that the
classical methods of enzyme separations used by
prcvious authors (Monod antl Colin, 1932;
I<uby and Lardy, 1353; Lester, 193) for the
purification of ~-galactosidasc (lid not satisfy
either of these criteria and could not bc uscd.
without drastic modification. Other methods
irere sought and pnrticulnr attcntion was prlid
to the potentialities of clcctrophorctic scpardon
Gf protein on filtcr paper (Durruni, 1950; Crcmcr
:ind Tisclius, 1950; Iiunlicl antl Tisclius, 1951).
Despite a considerable `amount of effort it was
{ound impossible to adapt this procedure for the

-.

    :it liml. `!`ltc :imount of 1nstcri:il wliirh
could 1:c nccomrmtlxtcvl in mc Ian, svcii tisiiig
pil(ss or p:~pcr &;is, W:IS too srixtll for fc:isiIJi\ity.
III :rclc!it,ion, t!ic resolution of tlrc ciuyme fl.orn
of,licr cnnt;imin::tin:: protcins was not sdhciently
nhnrp on nay fi!tcr paper types nvil:rGlc for trial.
tittention, thcrcforc, was turned to ot.iicr solit1
m3tcri:il which could act ns :I stiitnhlc ncirtral
support. ~Vliilc t!ic prcsent invcstig:ttions \yere
in progIws, K~inlid :ml Si:it.cr (In$>) rcportcd
liricily on :i~i cstcnsion of f,hc pxpcr strip rnc:tliacl.
Thy cnmc to the conclusion th;h stnrc:li XIS tlic
most t1csir:hlc ni:rtcri:il to USC, bnscrl h0t.h on
low atlsorbing c:Lpacity for protcin ant1 minimal
clcctroosmotic flow of wntcr. In agrecmcrit with
Kunkel and Slatcr (1953) thc pregcrit :iuthors
found that insoluble potato starch yicltlctl thc
best rcsults of all tlic matcrials tcstcd. It diowcd
no tcndcncy to adsorb spccifically cither the
/5'-g:rlactositlssc or thc other protcins prcsen t in
estracts of E. coli. This lnttcr property is of the
utmost impoitnnce if trailing of protcins in the
colunin is to bc avoirlcd.

Thc insolubic potato starch used \{-as obtniiietl
from E. Ti. Snrgcnt and Co. It \vas found ncccs-
snry to rernovc contnninsting nitrogcnous
.material from :JI conimcrcinl potato stnrc~ics cs-
amincd. This was accornplishctl by ~vndiing t.he
Shl'Ch scvcr:d tinits with thrcc tiIi>$s .its volunie
of 0.05 H dk:ili. ?'!IC :Lk:ili w:is r.rino\-cc! witli
water, which then was follonrtl by scvcr:il
washiigs with the buficr to be cniployed. Tiic
st:irch \vas stold under buficr in thc colcl at
4 C. Stnrcli for the solidification of inpiit nwtcrial
was obtaincd by drying starch following the
water wash.
Thc stc;)s followtl in the isolation of enzyme
nxry be siinirnnrizctl briefly as follows:
(1) Subsccjucnt to induction of enzyme, cells
are harvcstcd and wnshctl with €120 folloivcd by
centrilugntion. All the subsequent steps were
carried out at 4 C.

(2) Cells arc rcsuspcntlcd to a density of ap-
prosiinately 50 mg wct weight per ml and sub-
jected to sonic disintcgration in a 10 ki.locyclc
Raytheon oscillator (70 v at approximately one
ampcrc) for 15 to 20 min.
(3) Thc resulhnt extract is cciitrifuged for 45
minutes :it 15,200 C in a Sorvdl SS-1 ccntrifugc.
Thc pcllet is discarded.
61) The enzymc in the supernate of st.cp 3 is
concentrated by ammonium sulfate frnctionation

4.23

sulf:itc (52 p~r ccnt) prxipit:itioii rq)(::~td. 'l'I:c
sccontl 1)rccipit::tc is rcdiso!vcd iii If-0, :tntl tlic
[)TI is n&us:c~l to ncutrdity. In t!ik iii;in:icr, :in
incrcnsc 0: Iict\\.ci.ii fi~c :t~d tt!rifo!J in cnzyinc
co1iccnt:;ition can IIC :icliievctl alo:i:: witli the
rc~noval oi a higc portion of ttic 1ioii;)rotciii
n1atcri:il. Thc p1irific:ltion attni;lcd by tllcsc
stcps r:dy csccctls 3 X. Ncvcrtilc!css, such Con-
cci-ltrtltion stcps arc IICCCSS::~~ idicncvcr it is
(1cjir:ibIc ts 1)1:ic,c rcuson:ibly Inrgc uinounts oi
act.ivc cnzynic on thc columns.
(5) The residual ammonium sdfztc in thc
cnzymc solution which attcnds carrying out stcp
4 w:is removed by tl'inlysis against \voter. This
reniovnl \vas neccssary to avoid . tr;riliiig of
protcin during tlic course of tlic clcctropliorctic
sclwation.
(Gj In tlic prcscnt invcstigat,ion, starch
columns 5 rni in width gcncr:JIy \:.crc usctl in tlic
first i'uns. Tlic thick starch slurry nw pourcrl in
lucite troughs -lSxii_!ong arid 1.5 cm high.
Esccss nioisturc w:is icniovcd by strips of 1ic:tvy
blotting pnpcr. Tlic columns arc :illoivccl to sit in
contact with tlic elcctroclc \cds for 3 period of
two hour5 to pcrinit crpilibrtltion. Such colutuns
cm casily iiccoinniod:ttc 7 nil (cqrrivalcnt to
bctivccn 50 nnrl 70 in1 of origiii:d t:str:tct) of tho
tli:tl;.sc:tl cstl:i(:t:j u1)t:~iiiul in stci) 5 if tivo cin
trciidics :trc cut ,out iu the prcforntctl column
(closc to ncgptivc cell). Dry st:rrch is atldcd to tlic
protcin solution until 3 consistcncy is rcncliccl
w!iich is cqiiivalent to thnt of tlic colrann. Tlic
contents arc p1:iccd in tlic initiiil trcncli and
furthcr solidification achicvcd by adding niorc
dry starch. Thc columns thcn arc covcrcd with
lucite tops and placed bctwcen two clcctroclc
vesscls similar in construction to those described
by ICunkel and Tisclius (1931). Contxt betwcen
tlic eiids of the starch column and the clcctrodc
vessels is acconiplishcd by means cjf plastic
spongcs sitting in the forward compnrtiuents of
the vessels. The eniirc apparatus, other tlian the
constant voltage supply, is run in ;1 cold room
held at 4 C. EfEcient cooling of the colurnn is
effcctcd by having it. in contact, both froin nbove
.and below, with lead bricks such 3s arc com-
monly uscd for radioactive shielding. Thc voltage
applicd rangcd bctivecn 300 to 450 v yic!ding an
ampcrcge between 6 to 25 milliampercs, depend-

iy: 011 the c~~Jss-s~:(;t.iori:II :t:-e:i Cli t1:c (

-9 I . c hvc cil1;)!oyc(! routiiicly 0.iE.i 11 S3;I':i);.
iOi:,O (hicrdr) :IS tlic hit?cr :djwtctl tt, 1; ;:
dr.-ircd pi1 nt 4 C with IICi. This buricr   ! :
usccl convcnicnlly nt bctb p11 S.4 and pi!

TL~C colunln is uiiotd to ticvciop for ~)ct;..~,:,
12 io 1.: kr, by which tinic (:ti 3;O \..) ti;(; ,;.
gnlactosihc nil1 11:ivc movcd :;!iout 3.7 c:i: f:,.:,,
tiic starting pusition. Tlic position of tlic ciq1..:
pc:ik cxn bc c!ctcrminctl c:isily in :L vnricty ,,!
ways. Onc inctliod is to rcmovc saiiiplc p!qj :.;
one cm iiitcrvals along the colui~in with the ::i,!
:I trocw cut off near the nub ant1 fitted to
syringc. Tlic starch plugs so obtaincd then L:?
p!nccd in scpmitc tubcs and elutcd with lI,o f.:
buihr. Enzynle zwys and protcin dctci.:;li.
nations can be made then on tlic eluates. Ti,;
elution is cnrrictl out at 4 C for '10 riiiniitc> wi:i':
iUIj(1s arc Iicing aIi:Lkcri in an iiiciinctl positif):; :
a 1JhtfOlm sl:3kcr. 1VItr.n p1:icctl in aii tip:.- .
positon tho starcli ii1 t!lc td~s ecLt1c.s i*:iC,iii i-3
Iiiir:utcs, permitting thc t1cc:int:itio:i of [':
chute. Oncc tlie cnaymc 11:~s been located, ti..
block contzining the peak region can be cut I:!
and rcplaccd in a preparcd initial trcncli ci :
ircs?~ column. This proccss can be rcpcated I;:,:
coxkint specific. activity in tlic peak rqjrl:,' ;
att;Liiicd.
Conditions of inrhctiorr. Ikzyrnc was iIiilt;~~~ i
in :tcr;:tctl cxilturcs growing csponcnti:dl:; j..
synt.lii:tic mcdiiiin with i-l:ict:itc as the Y'
cnrbon and aicigy SOIIICC. Thc latter is "new..:
with rcspcct to &plactosidasc synthesis, in ;: r
scnsc of not intericring with it. Melibiose, c.!.i.'.
is not utiliznblc by strain ML, was uscd
principal inductor to avoid the comp!ic;!:i:.:.i
@lonod and Colin, 1952) which can atteritl ti .
presence of a nietabolizabIe inductor.

..

ti

EXPERIJIEXTAL RESULTS

Purijica?ion ~f enzyne. It is the purpose o! I:.
presciit scction to describe some reprcscl:t:a::. .
details obtained in the course of purifyin;: :
galactosidase iroin pnrti:dly induced cclL 0: i
coli. The rcintivc efficiency of the starch co!l;:. '
procedure may be illustrated by the resdij c'
scrved in the first run with n crudc estnrt ::
diich no attcmpt at prclirninzry purificuti[il ...
concentration of enzyme by nicnns of 3n:nioi
sulfate fractionation was attempted.
Lognrithniically growing cells, s~~p~D(!tf: ',
300 ml of synthctic medium, were incubd :'

c 90

,:i; C with acrntion in t!ic ~;rc,;c!icc of 0.601 31
U:p:i!)io.:c as inductor. TI:(: intlwtion uxs .:Aoppcd
:;fipr 97,000 units (niy:.; of o-i?itio!,lic;iyl-~-
!)-;::l:ictosidc split per miiiutc) of cnq
I:l!cn synthcsizcd. During this s:mc pe
:;icrc:isc in -ccll rnus cquiv:ht to 0.37 d

3118.1 subjccteil to sonic Igsis. Thc estrn
,.!cv:~~ zii of ccll debris by ccntrifugntio
Ili!tliitcs.stt.15,?00 G. The rcsdtiiig est
t!linctI ~O,~OO units of cnzynic per 111

est:xt was plnccd in n two cm wide stitrch
column in an initial trcnch about Oilc cni in
1Cngth. The column was allowcd to develop for
950 niin at 370 v and 11 millianipcrcs. Then it
\\.:is cut into one cn~ blocks, cnc!i of which was
cl:ltctl by shaking with 4 nil of I&O for one liou~.
'rhc cluntcs wcrc analyzed thcn for cnzymc ancl
protein. The rcsillts obitlinccl arc dcscrihd in
iigiirc 1 in whicli arc plottctl tlic units of enzyme
:ind pg of protcin pcr ml of clu:lte as n funciioii
oi tlt dishncc from the starting trcncl:. It will
\IC noted that a large amount of protcin in suh
cstrwts moves vcry slowly. Thc cvidciice nvail-
riblc indicntcs that much of t!iis SIOW moving
protein is associntcd with nuclcic acid. Thc cn-
zyme mows fairly mpidly nntl ns n rclntivcly
narrow pcak. The spccific activity of the uizy~nc
in thc pcdi rcgion is 300 2nd is betwccn 330 :ind
-io0 for the two cm on cithcr si& of tlic pm!;.
Tiic.;c values arc to be coniparctl with 13.2 for thc
input mntcrinl. Thus, one run cnn cficct nn np-
prosimntcly 30-fold purification. Eighty pcr ccnt
of thc input enzyme can be rccovcrcd readily in
any given run.
The cffect of repeated succcssivc runs on the
purification of the cnzynie mny now be csmiiucd.
Cells ivere induced to form cnzyme much as in tine
condition described above. hhlbiose (0.001
11) was the~indu~tor;-zrid the ceKs were growing
lognrithniically for a pcriod equivalent to one
division during the induction.
Estracts mcrc prcpAred by sonic disintcgrntion
as'dcscribed -previously, and aftcf rcmov:il of cell
dcbris, the enzymc was pnrtinlly purified and
concci1tr;rtcd Ly tho :tiiiinoiiiuni solf:rtc fmc-
tionntion procctlurc dct.:iilctl nntlcr Mcthotls.
Thc initial trcnch accomniod:~tcd 4.5 nil of thc
conccntrntcd cnzynic prcpar:ition. Thc ColIiinn
\vas allowcd to develop for 1,OOS ininutcs 3t 245
v and 15 ms. The cuzyme pcnk was 1oc::tcd and

-EhZYUE

\-

s!)t?cific activity of' 13.2. one in1 of the nbove

DlSiANCi Ih CII

-. .

Figure 1. DistrihuLion of enzynic and protein
dong the starch coliimn in a first elcctrophorctic
run nt 370 v and 11 riiillinmpcrcs for QSO minutcs.
At thc pli cmployctl (8.5) thc enzymc travels
towards positive electrode.

t1:c spccific activity of thc ciizyrnc in tlic pcak
rcgion detcrniincd by cximining il $2 cnl strip
rcmovcd from thc column. Oncc locatctl, the
pcnk rcgion of enzyme in thc starch column \vas
cut out, plnccd into an initial trcncli oi ap-
proprintc size in a frcsh column, and the process
repented. Tnblc 1 sunirnarizes thc results obtaincd
in four succcssive runs. It will be noted thh the
spccific activity of tlic initial prepnration was -15
and th:it the first run gnvc an cnzymc prcpn-
ration at tlic pc:ili with n specific activity of 976
corresponding to n 20-fold incrcnsc. As is in-
dicaicd by thc rcsriits of run no. -1, tlic mnsimal
puri5cni.ion wns ncliievcd by run no. 3..
Unijorniily of prokin labeling. The prirposc of
thc prcssnt cspcrimcnts rcquircd comparison
bctnccn the spccific radioactivity of thc cnzymc
and the nonenzyrnztic protcin in the ccll. Such
a coinpnrison is nicmingful only if the non-
enzyrnntic protcins arc uniformly labclcd. Initial
uniformity in the labcling is assured rcadily by
employirg an inoculum which is small relative to
the End yield of culture n-hen growing up cells in
the C1,L-l:ict,$c. The :issumption th:it growth of
such cclb in an identical mcdiuni which vas un-
1:ibcicd ~vvould rcsult in 3 .unifornl dilution of ,211
csistcnt protcins \vcs tested directly. Cclls
tinifoin:ly Iabcled to thc cstellt of 101 coulits
pr inin pcr pg N wrc p~~p:ird by gro\vth iu
C'~'-lnct.:lLc. 'i'hc cclls \vcrc wnshctl tlroroll~hly
wi'iii cold mcdium cont:liniiig 0.3 pcr ccnt of
ut1 lnljclcd L-lactatc, rcsuspcndcd in tlic sxnc
n~edium, ancl a1loivc:tl to go through npprosi-
mately one divisiou. The cclls then wcre hnr-

42.1 *

YILLIA::PLRLS

I

PtiS t.0. DUPATlOS I VOLTS

I--

I

?I.:,, I

Initial prcparntion

1 15.0
                  2 :ci 15.5
                   245 13.0

lG.5

2 1,059
3
4

vcstcd, wnslicd, and lysctl by sonic ticatrncrit.
Aftcr rcinovnl of ccll debris by ccntrifugx'iion,
and witliout :my prior friiction::tion, tlic cxtract
was placed in t.he starch column :ml :i!lowcd to
dcvclop at 400 v ant1 11 iailliaupcrcs for a pcriod
of about 15 hr. Subscqucntly, thc column was cut
up into portions one cm in length, and each
section eluted separately. The proteins in thc
eluates mere precipitated and purificd by the
mcthods dcscribcd, and the spccific radio-
activities dctcrniincd. Thc mcan spccific radio-
activity c,f t,lic protein (plus and minus two
st:intl:trtl dcviations) of 30 such sinrc:h scctio:is
 is found to Ix 4A.2 + 4.3 counts 1)cr rnin per
pg S. All the \~:~ucs \Ycie ciicoiii1)asscd within
tcvo st:indard tlcvintions of thc nicm i:itiicating
no stntistic:tl dificrcncc in tlic dcgrcc of dilution
cspcrienccd by the proteins which were so
isolated and csnniincd.

Tire niuxi?irul spccijc E?L:!/I)LC activil:j. D:it:L ob-
tained in tiic cour~c of rcpcatcd purification of
enzyme on tlic starc11 colunins suggcstcd that the
niasiinal specific activity of thc enzynic was in
the ncigliborliood of 1,900 nip31 pcr niin per
pg N undcr the conditions of our assny. Com-
pnrison with prcviously published (Cohn and,
Monocl, 1951; Lcstcr, 195'2; I<uby 2nd Lardy,
1333) figurcs on specific activitics indicntcs th:it
tlio sttircli column 1)rocctlurc yiclds piqxu'ntions
of high purity. It w:is of intcrcst, p:utkiil:irly in
vicw of its uscfulncss :is .:I. ~iic':iiis oC cstiniating
enziinc protein, to obtain further infoimation on
the validity of thc maximal figurc. The usc of
specific precipitation of radioactively labcled

enzyme uith purificil antiscruni suy,ptcrl its&
as I possible mcthoti for :inotlier check on tihi,
figurc. Cohn and Torri:tni (1952) 3nd Cohx
(1933), have provitlcd the conditions undcr which
such prccipit3tions cnn be carried out. They li:Lve
dcnionstratctl also that the enzyme in the In-
soluble antigcn-antibody coniplcx exprcsscs fail
activity. Werc the enzyme in such a coinplcs
uniformly labclcd with a radioactive tracer 3Ril
its specific radioactivity (in counts per niin pei
pg K) known, tlic amount of enzyme protein in
thc prccipitatc could bc tlctcrmined rcatlily.
IVitl; the httcr figwe and thc cnzyinc activity,
tlic s;iccific cnzynic activity wou!tl bc known.
Tiic efficiency of such spcciSc prccipitation in
rcnioving cstraiicous mntcrial is illustratcct by th~
work of Cohn and Ilogness (Xonoti and Cbh,
1923) 3s \\.ell as the results reported in the prescnt
paper.
Uniformly Inbclcd cck wcre grown with am-
tion in s~mthetic nicdiuni containing C"-L-
lactate as ilic solc carbon source. During thc
growth, mclibiosc (0.001 ai) was introduced
an inductor. .Such conditions would insure uni-
formity of 1:ibeling of all tlie proteins of the ccll,
including the P-gal:xtosicla3c. The cells were
hnrvcsted, estracts prcparcd, and enzrme con-
ccntrntcd 3s prcviously dcscribcd. An aliqriot wns
plnccd on a starch coluinn niitl :rllowcd to tlc\-clop.
Tilc cnzynic pcak \ws locatcd :md rc-nrn in :L
frcsh column, The rcmairtdcr of thc first coluinn
\vas cut up into 'one cm sections which were
eluted, and the nvcrngc 'spccific radioactivity of
the proteins determined. The mean &2 stmdd

errors IWS found to bc 15s i ;;.!) ccjiiiits per inin

pee;: Y, :And no significiint clcvi;\tiwi.< fro:n it
\Vfl'C ol~scrvctl. Xftc; sc\cr:t1 dU
tile ~hrcli columns t!ic ciuyir.c
c!utcJ. The cluatc contAincti 5S3 cnzyiiic units
pes ml.

Onc nil of this c1unt.c ws subjcctccl to prc-
eipitntion with 0-gnixtosii  c mlixsiim puri-
fied and prepared :is c!cscri!irtl ui~dcr I\.lctliocls.

-Q&II;K~C thc spccifcity of thc prccipit:ition it
\vas cnrlluil.'out. in the prcscace of un1:ibclcd
cstrnct from noninducctl cclls, which insures tlint
fill noncnzyiixitic antigciiic con-iponcnts \voulcl IIC
in t,l~ region of antigcn csccss. The rcnction
mixture had thc following componcnts: 1 nil of
eluate, 1 ml of nntiscrurn, 1 in1 of unlabclcd
noninducccl estrxt, 0.2 nil of 10 pcr ccnt KaC1,
0.2 nil of pII 7.5 p1iosph:rtc buficr. Tlic nntiserum
\vas nddcd last. Tlic rcnctioii misturc n':ts hcld for
96 hours at 4 C, and tlic prucipltatc collcctctl by
centrifugation and w:islieci thrcc tinics with
cold 0.02 31 phosphntc buffer at pH 7.0. The
precipitate N~S rcsuspcndcd then with honio-
gciiizntion in 0.9 ml of tlie same buffcr.

Both the supernate ant1 the rcsuspcndcd pre-
cipitate werc assnycd for cnzynie. Xonc was found
in tlic fornier, and 06 pcr cent of thc input enzyme
wtivity xis accounted for in the antigcn-anti-
body precipitate.
Rndionctive counts on a 0.4 in1 n!iquot of the
rcsuspcndeci prccipitntc yiclclcd a Kiluc of 49.8
ct per nil. From the nvcrage spccific r:idioactivity
of the cellular protein wc find tlint t!ic :mtigcn-
antibody complcs contains 0.32 pg N of ccllu-
Inr protein per ml. We find, assuming that all
the input enzyrm mitcrid is in t?ic coniplcx,
that the specific ciizynic activity is 1,820 nipx pcr
min pcr pg N, in reasonable agrccmcnt with the
prcvious "niasinial" figure of 1,900.
Synthesis of enzpe in labelcd cclls suspcnded in
iciilabelcd -7ncdiuz1~. _Uniformly Iabclcd cella of E.
coli were obtained by growtli of a cuilure in one
liter of synthetic medium containing 0.1 pcr
cent L-lactic acid with a spccific radioactivity of
-1.6 x.1O3-ct per min pcr px The culture IUS
started by n small inoculum from :I stock slunt
and allowcd to incubate with vigorous ncrntion
3t 37 c for 16 110Ul.S. TlkC CC!h \\'I.!I'~! h:ll'VCiitCtl
by. ccntrifugntion :ind i~flslicd twicc with colt1
mcdium to rciiiovc rcsic!unI r:r(lio:ictivity nnd rc-
suspcntlcd in 5 In1 of I-120. 'I'hc 1:rttcr tlicn wis
added to 4 liters of synthctic iiicdium previously

ec~uilibrcltcd to 37 C atid containing 0.2 pcr ccnt
of unl~bcled L-l::cliic acid 3s t!ic solc carbon
so~ir(:c. Tlic siiqxi.?ion \vas incuixttctl with
vigoroils acrntion :it 37 C. S:intplc.i wxc rcniovul
at 10 inin intcrv:rls for opticnl dciisity tlttcrini-
i;citions to asccrtnin thc tinic of (JnSct of loyarith-
rnic growth. Culturcs .SO tre:itccl rarciy liave lag
pcriods escccding 10 minutcs. Intluctor in the
form of melibiose (0.001 ai) ivxj nddcd nftcr
Icgarithmic growth \vas :immd and the incuba-
tion continued iri tlic prcscncc of inductor for a
period corrcsponding to 0.9 divisions. P:iinplcs
wcrc rcmovcd pcriot1ic:iily for Loth optical
dcnsity clctcrmiriatioris arid enaymc :issay.
At the cnd of tlie period indicatcd, tltc cells
WC~C liarvestctl in a Sliarplcs centrifuge, w:ishcd
with 90 nil of H,O, rfsuspcndctl in 50 nil of 1-120,
and sonicatcd. The enzyme in the cstrnct was
partially purificrl and conccntrated by :tmmoniuni
su1f:rtc fractionation ;is tlcscribcd prcviously, and
fu;thcr purification achicvcd on 3 starch colunin.
Table 2 dcscribcs thc progress of the purification
during succcssivc ru~is of the pcnk region. This
particular cspcrinient was tcrminatctl aftcr the
fourth run since therc \vas not enough nintcrinl
left to pcrniit adcquatc cstiniations of protein
content, cneyme, nnd radionctivity in n'fifth run.
Tiic data arc given in terms of tlie spccific cnzv-
niatic nctivity arid specific radioactivity of tlic
pcdc region. The latter is compared with the
spccific mdioactivity of the ,zvcr:igc protcin.
it is evident from an esaminstion of tlic dntn
thnt as tlic purification oi tlic enzynlc progresses
thc specific radio:ictivity falls. Tlic ():ita dctnilcd
in t:tlh 2 support titc stntcnicnt that less t1t:tn
9.1 per cent of the carbon of the newly formed
eiizymc molecules can be derived from any prc-
esistirtg carbon in tlic cell.
In other similar espcrimcnts, analogous results
werc obtaincd, the dcgrec to which tlic cneyme
could be frced of radioxtivity being about the
snine and indcpcntlcnt of the Icngth of the in-
iluction..Thc purific:ition and nnnlyticnl methods
ci-nploycd dcniand n niinininl :iniou:it of cnzynic
lor the succcssful completion of the csperinient.
This lowcr liniit is such as to precldc espcri-
nicnt involving inductions, running much below
0,s divisions, oven with tho USC of tho inoro
powcrful inductors such as n-butyl-P-D-gn-
lnc tosidc.
Tlic rlucstioii ariscs wlictlicr thc some\\-li:it Icss
th311 10 pcr ccnt r:idionctiw carbon found in the

' enzyme purificd by t!lc stnrcli column nictllod
rcprcsciits n sni:~!\, !)ut nic:isw:i!)ic, proportionntc
utiliz:ition of prccsisting cnrLon in tic coiirsc of
enzyme syiitI1c:jis. Infoiniition pcrti:ient to t!iis
issue nisy bc Qbtnincd by conip:Lring columns .I
nnd G of ttlbie 2. Column 4 rcpi'cscnts thc "per-
centage purificntion" nchievcd ct each step cal-
culzted froni the specific cnzync activity ns
compared rrith thc mnsimal value of 1,900.
Colutnn G cslcuktcs tlic "pcrcciitngc puri5c:r:
tion" from thc rclativc yi)ccific rndianctivity
(column 5) on tlic nssumptioii tht tlic cnzymc is
com!~lctcly.~:tLclcd. Thc I,cl:t-iivc!); pot1 :L~I.CC-
mcnt betwccn tlicsc figurcs sriggcsts t,liat the
rcsirliul r:itlio:ictivity is not cl;zyni:itic but is
lather ;~ss:ig:inblc to contaniin:itin,- nia.tcri:il.
Such cdculations, hovxvcr, :trc not of sufiicicnt
scnsitivity to IIC c(Jllq)!ctdy dccisivc ;it low
I'evcls. TIici,cforc, imniunoIo!;ic31 prccipit:rtion
\vas usctl to dccitlc this question.

Spccijc. prccigiihtion oj particilly purijicd
enzyme with (oi~isci-1m2. Tlic immunologic pro-
ccdures dcscribcd above were usctl. It may be
riotctl that it WLS foiincl, in agrccnicnt with
Cohn and Monod (;>ersonal comniunicntion),
that sclective removal of cnzymc protcin wit!i
nntiscwni cannot be ncliievetl in crude cstrncts.
Prelin-inary partin1 purification is essential. In .

>I mclibiosc. The prcpnr:ttion of the extract ;,,.. .
placed in the starch column, and the pcdi :r
of cnajmc activity run thrce succe.+nive tinm :.
st:irch columns. On the third run, tlic c1u:cte irp:,
the enzyme pcxk region contained a protrin i\ii.,
a specific enzpiatic activity of 1,300 mp,[ p-
mill per pg N. Comparison with t.he rnxi!;. .:
spccific enzynib activity would indicate :li::t :
pcr ccnt of purity hnd bccc nciiicvctl. l'h I:.;
is ii1 csccllclit ag,:.ccrnCi1t \\.\.it11 ~11::t V:OC~: :
estirnrLtcd from its spcciiic I.:dioxiivitp, ~:i:
ing thc cnayix p:otc!n vas i:nLi)ckii. 7';
protcixi conlnir:ctl 1:iIwl to t,lic c~tc~it oi 111 :
et pcr rnin per. pg S which is 30.1 pcr cciit ni i!;
value of 63.7 ct pci. min pcr pg K iountl for I!.
nvcmgc noncnzynintic protein in tlic mnc ci!.

The ch:itc, contnining 5,000 units of ciiz;;T-
ptx id, wis subjcctctl to spccific prccipitnti.:
with purified &plnctositl:isc antiscrrim ioilmi: :
the proccdurc t1escrii)ctl ;I~CJVC. Ag:iin to iixi.1
thc specificity of the prccipitatioii it \vas c3rrk :
out in the prcscnce of un1:ibclctl extrxt fits:..
noninduccd cells, so that all noncnzyni:itic cc..
poncnts would be in the region of :mtigcn cw:
The reaction niisturc was hcld at 4 C ki 4.
hours, and the precipitate n-ns collected 11:
centrifugation. It was tvashed tnice witil 0.1 2
yhosphxte buffer at 7.5 and rcsuspciided it?,!.

the present esi>crimcnt this prcli1nin:iry puri-  honiogcnization in 0.G nil of H20. An aliqtat (!
fic;rtion \v:is :~tt:lincd by tlic st:ircii coliimn 0.4 1111 \r:is used far the dctcrniin:tt.ion of rdii
mctliotl. C" labclctl wlls WCI'C pi~q):~rcd nnd activity :~nd 0.05 nil for the :LSP:LJ' of cnz~n:?
inducctl to. form cnzynx while suspcntlcd in  All of the origin31 activity contnincd in ti.:
unlabclcd lactntc nictliuni as dcscribcd &GW.  eluate yns accounted for in the nntigcn-mtik.1:
The induction \vas carried out in log phasc for a  prccipitntc, which checked with the fnct tlut r.
period of 0.5 doublings in the prcscncc of 0.001  detcctdh enaynie \vas found in the supermi<

TADLE 3

p~ri]h~!iori by spccijic prcc;j,it(;;io:i i~ith un!iscrtotL
  Oiic nil of stnrcli column t\li~::tc: .,\.:is :t[itlcd to thc
[dlo\ving: 1.0 nil of tiiil:tI~c!c~~ cstr;rct from
Iloiiintluccd cclls, 0.2 nil oi IO 2cr cent S:rCI;
0.2 nit of 0.1 11 phosph:itc bii:fcr nt I)iI i.5. To tlic
rc,iiiltiiig inisturc v;:is :dclutl 1 iii1 or purificd
!,:Itiscruni. Tfic rcsu!tin;: j:rcci;)it:ltc wts xnslicd
:,ilti IircpLrctl ns tlcscril,ci! in tiic test. The :iliquot
lllc:isiird io: r:,tlionctivity corit ni:iccl 0.33 X 10'
\,i.pncunits. Thc counting ivm cGiiriiincd until
5,910 coiints-.fid nccuniul:itcd (300 niin). 11
h:lckground count ovcr thc sniiic tinic pcriod
vicldcd 5,972 counts. Thc nbsolutc orror wzs
&tcrniincd froni tk.c rc1:irion 12 = IidX`t + xs
ivhcrc Kn is the background count, Xs the count
of thc s:nnplc, and I; thc prob:rbiliry constant.
Tlic latter was taken us 3.20, which yields the
*`n99/1,0oO'' error. Thc protcin nssignnble to
cr:xyn:e in tlic an ti gc n -:I titi body prcc i pi tn tc was
c!ctcriiiinctl from thc ninsinid cnxyn:c spccific
r,ctivity value of 1,000 and thc obscrved cnzynic
sctivity. The figurcs in the second row are cd-
culatcd fron thc latter ai;d the dcicrrnincd
absolute error of counting. The ch:rnccs are one
in a thousand that thc actual figures can Lc

..__ .-

greater than those stated. --

Bcforc precipitation
\sitii antiserain.. . .
Aftcr precipitation
with antiserum.. ..

-I-!

176 1 19.2 1 30
<3.6 , <O.GO' <1.0

I`

I

fornix1 cnzymc mo!cculc conies iruiii 3ny prc-
csisting carbon in the cell.

DIbc`usblos

c1

I he cs:mhcnts dcscriherl in the p;e:icnt ppcr
oiier iittlc support fur t11c prccsistcnce in tllc ccil
of nny significant aniwiiit of prcciii.:or ni:itcii:tI,
simple or complcs, rvhich is utiiiz:Afe for the
synthcsis of ncw ciizynic molcculcs. Thcsc :~.,u!t.i,
taken together wi tli the indcpcncicnt!y pcrformcd
cspcrimcnts of Cohn and Ilogncss (lloixd ant1
Colin, 1053), make it diflicult to avoid the cw-
elusion that the induced sgnt1icsi.i of fl-g:l-
lactosiclase is a virtu:tlly de nouo ~GCCSS eni-
ploying principally amino acids \vhicli arc formed
suLscquent to thc monicnt of the addition of the
inclucc1*.
A brief statement may be intcrposcd here
concerning protcin turnover. It is obvious tliat
had there been any estensive brcakdot\-n nnd
rcsynthcsis of protcin in the course of the cs-
periincnts reported here, a proportionnte aniou:it
of 1:tbcl would hnvc ninde its nppearance in tt;e
P-galactosidase. No cvidcnce was dctectctl ior
such burnover. The snnie is true for the csperi-
nicnts of Cohn and I-Iogncss dcscribed by blono:i
and Cohn . (1953); In tlic latter csperinients
attenipts were made to see whether unlabc!ed
eriapie in S35 lnbcled cclls would pic!; Up label
either in the prcscnce or in t!ic abscncc`of in-
ducer. No such interchange was oliserved.
Such oimxvations would appear to pc)sc the
problem of whether the protciris of growing
bactcrial cclls differ from those of adult mnm-
malinn tissue in which "turnovcr" can be cfe-
tccted. In part the difierence is a spurious one,
assignable to the markedly different time scales
of the cspcrimcnts performable with mice anti
nien as compared with E. coli. A sample calcu-
lation should make the relevant issue clcnrer.
We may take 3 half-life of 6 days (Borsook,
19.53) :is a sufficiently reprcscntativc figure for
tlie turnclver rate of a rc1:itidy rLipidly syn-
tliwiziiig mammalian tissue. This xvould yield 3
tlccay constant in hours of  -0.0048. A com-
parntivcly long term esperiment with E. coli,
of the typc wc'hnve dcscribed, u-ould extend over
a pcriod of two hours. Coiisidcr otic liter cif un
E. coli culturc :it 1 x lo9 orgxiisnis per nil
uiiformly Iabclctl to a lcvcl of 100 counts per
niiri per pg protein N. If these protcins possessed
the decay constant noted above, they would, in

tIic c0~i.i~' of .z tiro iiwr i  (1, 1iiicr:rtc oilc :E:.
cent oi ilicir fiiiiir);ic;i.<i!,;:, o:' 1 x 10` ct. 1)ci miii.
Assuming :is scciiis :c~soiialric, tii:tt ;t!I ~rrtrtcins
bcing synt1icsizil:l (xi 1iu:icipatc cqwiiy in
in corpora t i ng tii is rdi 03 ct i vi t:, and  t 11:: t 0.1
?cr cent of the protcia 1)eiliK iornicd in thc two
hour inc!uction pcriod is ,3-g:nl:ictosidise, npprosi-
m3tcIy 10 ct pcr niin \vouid bc iound in ilic entire
enzyme fr:tct,ion. This wo;iId be bxely c!etcctablc
evcn if a consiricr:tli!c po:tion of tlic cnzpc
fornm! w:\s isohtcd in :I prc shtc. On thc basis
of spccific r:tciio:ictivity, it nould bc just Lclo\\,
the lavul of ndaquatc mcasi:rcmcnt since it \.;auld
be somewhat lcss tlinn oiic ct pcr inin per pg X.

It is clear from tlic d~ve tliscussio:~ that es-
pcriments on intlucctl syntlicsis, performed in
thc niaiincr tlcscribctl, niiglit well iiiiss ttii.novcr
r:itcs oi Icvds c1iar:ictcris:tic of ~nanim:ilinn
tiswcs. The tl:rtn nccuiiiu1::tcd in tlic coursc of tiic
prcscnt investigation ~vortfd indicate th:tt the
protein brenkdown rates in growing E. coli cclls
cannot have t!ecsy constsnts much greater thnn
0.005, and they may possibly be lcss. This is in
goad agcemcnt with sonic recent results ob-
tained by Anlrer (1954, personal communicn-
tion) who followed the rc:casc of C%rginine
froni bncterial prot,cins in E. coli growing in
chcniostat. Thc rcsults inchtc that Icss than
onc pcr cent of tlic argininc iri protein w:is Ie-
lcasctl over n pxiotl corrcspoiiding to n 3.l-fold
incrc.sc in mass during &lie growth of nn E. coli
strain. hdOlSky (103j reports n sonicidiat
hig!icr figure for E. coli groiviiig in a syutlictic
medium under sonicwhat diKcrcnt conditions.
He finds that over a 30 hour pcriocl S per cent of
the labeled arginine is lost, corresponding to a

One further fact m:iy be noted. A simple c:d-
culatioi reveals that tlic specific rate of protein
synthesis (mnss/niass/unit timc) is of the order
1,000 timcs faster in bncterinl cclls growing in log
phase than that found, c.z., in tlic liver of the
adult rat in nitrogen balxicc, tlic proteins of
which have a half-Iifc of about G dnya. We 1iavc
scen nevertlic!e$s that the decry rate of bacterid
proteins cannot be much greater tlian that ,cor-
responding to a G d:ry half-life and is indecd,in
311 probability less. This ~vould appear to climi-
nnte any interprct:ition of "turnover", or the
"dynamic state" of proteins which seeks to link
in n compulsory n~anner rstes of protein break-

,decay constant of 0.00s.

kkj)crimcnt.s II:LYC lrccn tlcsigncd to test tilc
folioiving two possiijilitics for the sjmthcsis oj
P-gn1:rctosidn.w by Bsclrcricirirc coli:

(a) I'recursor -t free amino acids -, Active

cnzyrnc.
(b) Free nmino ncitls -+ Xctivc cnzymc.
Enzyme synthesis wis intlticcd in cclls u:]:~

fornily 1:rticlctl witli C;'` wiiilc ilicy w:'c pc;.
pcntIctl ill unlalxlctl nictliuin. 1sol:ltion nl,,!
purificntion of thc i3-gaI:xtodasc syntl,c.+.;:
revcnlcd tlint Icss tlm onc pcr cent of its cart,oll
could haw bccn dcrived from any ccllulnr com.
poncnts esisting prior to tlic moment 0: tilt
nrltlition of thc intluccr. Thcsc findings viitii:i!!y
c1iniiii:itc any Ilypotlicsis w11icIi ~~CSUJJ;~~SC,S tile
preexistence of j)rccur.wr ;n:ltcrial, siniple or
complex, convertihlc into cr:zyrnc.
Thc (lata further indicate that protcin ccp
thesis in the growing E. coli cell is virtually
irreversible. Prokin "turnover" evcn remotel!
approsimating the synthctic rate could not be
detected. I

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