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JOURNAL OF BACTERIOLOGYVol. 87, No. 5, pp. 1027-1033 May,
1964Copyright © 1964 by the American Society for Microbiology
Printed in U.S.A.
CONTROL OF ASPARTATE TRANSCARBAMYLASE ACTIVITY INTYPE 5
ADENOVIRUS-INFECTED HELA CELLS
RICHARD A. CONSIGLI' AND HAROLD S. GINSBERGDepartment of
Microbiology, School of Medicine, University of Pennsylvania,
Philadelphia, Pennsylvania
Received for publication 14 November 1963
ABSTRACT
CONSIGLI, RICHARD A. (University of Pennsyl-vania,
Philadelphia), AND HAROLD S. GINSBERG.Control of aspartate
transcarbamylase activityin type 5 adenovirus-infected HeLa cells.
J. Bacte-riol. 87:1027-1033. 1964.-Type 5 adenovirus infec-tion
induces increased aspartate transcarbamylase(ATCase) activity
during the period of magnifiednucleic acid biosynthesis. Increased
activity can beprevented by addition of pyrimidines to the cul-ture
medium. ATCase in HeLa cells is regulatedby feedback inhibition,
and purified enzyme canbe inhibited in vitro by cytidine
triphosphate(CTP). The enzyme from infected cells has a pHoptimum,
maximal velocity, and Km for aspar-tate distinctly different from
ATCase from controlcells. However, heating of ATCase from
uninfectedcells converts the enzyme so that its characteris-tics
are identical with enzyme from infected cells.Conversely, addition
of CTP to ATCase frominfected cells changes the characteristics of
theenzyme so that they are the same as those of en-zyme from
uninfected cells. The evidence pre-sented suggests that increased
nucleic acid bio-synthesis in infected cells initiates a release
fromfeedback inhibition and increases ATCase activityby reducing
the concentration of pyrimidines andpurines in the acid-soluble
pool.
Aspartate transcarbamylase (ATCase) activityincreases
approximately 10 hr after infection andreaches maximal activity 18
hr after type 5adenovirus infection of HeLa cells (Consigli
andGinsberg, 1964). The increased ATCase activity isdetectable at
approximately the time that bio-synthesis of virus-precursor
deoxyribonucleicacid (DNA) commences (Ginsberg and Dixon,1959,
1961; Flanagan and Ginsberg, 1962), andis dependent upon DNA and
protein synthesis(Consigli and Ginsberg, 1964). A comparison ofthe
characteristics of ATCase from uninfected andinfected HeLa cells
indicated that enzyme frominfected cells had an altered pH optimum,
in-
1 Pennsylvania Plan Scholar.
creased maximal velocity, and increased Km valuefor
aspartate.
Bacteria control biosynthesis of pyrimidines,purines, and other
metabolites for the physio-logical needs of the cell by means of
efficientmechanisms of feedback and enzyme repression(Gots, 1950,
1957; Love and Gots, 1955; Um-barger, 1956; Yates and Pardee,
1956a, b, 1957;Magasanik, 1957; Gorini and Maas, 1957; Gotsand
Gollub, 1959; Gerhart and Pardee, 1962).Similar controls have been
demonstrated inseveral mammalian organs (Sartorelli and LePage,
1958; Wylngaarden, Silberman, and Sadler,1958; Wyngaarden aild
Ashton, 1959; McFalland Magasanik, 1960; Le Page and Jones,
1961;Henderson, 1962; Schimke. 1962; Bresnick, 1962),and it was
predicted that ATCase activity inHeLa cells was regulated by
similar mechanisms.It was the objective of the study reported
inthis communication to investigate the control ofATCase in HeLa
cells and the effect of adenovirusinfection on the control
mechanisms. The data tobe reported indicate that ATCase in HeLa
cellsis under feedback control, that type 5 adenovirusinfection by
inducing increased biosynthesis ofnucleic acid removes the feedback
inhibition, andthat ATCase relieved of inhibition has
charac-teristics different from enzyme from uninfectedcells.
MATERIALS AND METHODS
The methods employed for tissue culture,propagation of virus,
virus infectivity titrations,preparation of ATCase, and assay of
enzyme wereidentical with those described in the accompany-ing
paper (Consigli and Ginsberg, 1964). Whenenzyme was utilized for
experiments on controlmechanisms, all solutions mixed with
enzymecontained 10-3 M mercaptoethanol and 10-4 Mneutral
ethylenediaminetetraacetic acid (EDTA)to protect active sites of
the enzyme from reactingwith trace heavy metals.
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CONSIGLI AND GINSBERG
Chenmicals. All chemicals were obtained com-mercially from
Calbiochem.
Acid-soluble pool analysis. Minimal mediumwas added to HeLa cell
monolayers, and cultureswere infected with type 5 adenovirus. The
cellswere harvested 18 hr after infection and washedonce with
phosphate-buffered saline (PBS).Appropriate samples were removed
for cellenumeration. Assays were performed on 5 X 108to 7 X 101
cells. Uninfected cultures werehandled identically for comparison
purposes. Thewashed cells were extracted with 5% perchloricacid
(PCA) for 30 min at 0 C. This lysate wascentrifuged at 1,510 X g
for 30 min at 4 C,and the supernatant fluid was saved. The
sedi-ment was washed once with 5% PCA and re-centrifuged, and the
supernatant fluids werepooled. The cold PCA pooled extract was
ad-justed to pH 2 to 4 by addition of KOH. Thequantity of purines,
pyrimidines, and theirderivatives was estimated from the
opticaldensity at 260 m,u of the cold PCA extract,assuming a molar
extinction coefficient of 10,000.These compounds were adsorbed onto
acid-
TABLE 1. Suppression of aspartate transcarbamyl-ase activity by
various pyrimidine nucleosides
Pyrimidine nucleoside 'tuLi Ureidosuccinateadded ~~Type of cells
(jsmoles synthe-Pyrimidinddedcleoside Type of cells sized per 2
X
106 cells*)
None (control) Infectedt 0.369Uninfected 0.171
Cytidine4 Infected 0.182Uninfected 0.188
Thymidinet Infected 0.165Uninfected 0.172
Uridinet Infected 0.156Uninfected 0.189
* The reaction mixture contained: cell-freelysate from 2 X 106
cells; aspartate (Na salt),2.0 X 10-2 M; carbamyl phosphate (Li
salt), 1.5 X10-2 M; tris-acetate buffer (pH 8.5), 0.04 M;
in-cubated 60 min at 35 C. The reaction was termin-ated by adding
0.5 ml of 10% trichloroacetic acid,and concentration of
ureidosuccinate synthesizedwas determined.
t Infected with 8 to 10 IDs0 per cell and incu-bated at 36 C for
18 hr.
t Concentration: 10-1 Mu. Added at time of in-fection.
washed Norit A (40 mg/,umole of base) overnightwith shaking.
Elution was performed by use ofdilute ethanolic ammonia (40%
ethanol, 1%concentrated NH3, 59% water). The charcoalwas eluted
four times, and the combined eluateswere taken to dryness; the
residue was hydro-lyzed with concentrated (88%) formic acid in
asealed thick-walled tube at 172 C for 45 min.This hydrolysate was
taken to dryness, and re-dissolved in 0.2 ml of 1 N HCl. The
hydrolysate(0.1 ml) was spotted on Whatman N 1 paper,and the bases
were separated by two-dimen-sional paper chromatography. The first
dimensionwas developed in methanol-HCl solvent (meth-anol, 140 ml;
concentrated HCl, 40 ml; and water,20 ml). The second-dimension
solvent wasisopropanol-NH3 [isopropanol, 85 ml; ammoniumhydroxide
(28%), 1.3 ml; and water, 15 ml].The bases were eluted from the
paper with 0.1N HCl. Their identity was established, and
theirconcentration was determined spectrophoto-metrically. The data
are expressed as mimolesper 106 cells. Bases that migrated (second
di-mension) in the known uracil-thvmine regionwere eluted and
rechromatographed for furtherseparation with a third solvent, ethyl
acetate-formate (ethyl acetate, 60 ml; formate, 5 ml; andwater, 35
ml), as the upper phase. These baseswere eluted and identified by
spectrophotometricanalysis and microbiological assay with
Escher-ichia coli mutants 15T- (thymine) and WC-(uracil; obtained
from S. S. Cohen, Departmentof Biochemistry, University of
Pennsylvania).
RESULTS
Effect of pyrimidine nucleosides on A TCase ac-tivity in type 5
adenovirus-infected cells. Experi-ments were planned to determine
whetherATCase in HeLa cells was controlled by end prod-ucts of the
pyrimidine pathway as had been dem-onstrated in bacteria by Yates
and Pardee (1956a,b, 1957). The increased enzyme activity in
ade-novirus-infected cells offered a suitable modelfor this
investigation. Pyrimidine nucleosides(10-5 M) were added to the
minimal maintenancemedium of cultures at the time of virus
infec-tion, and ATCase activity was assayed 18 hr afterinfection.
Results from two experiments (Table 1)indicate that the increase of
ATCase activity ininfected cells was suppressed by any of the
py-rimidine nucleosides employed. In contrast, thepyrimidine
nucleosides did not alter the level of
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CONTROL OF ASPARTATE TRANSCARBAMYLASE
enzyme activity in uninfected cells. It shouldalso be noted
that, although the pyrimidines in-hibited the increase in ATCase
activity in in-fected cells, virus multiplication was not
signifi-cantly reduced under these conditions.
Effect of nucleoside phosphates on ATCase invitro. That
pyrimidines can inhibit increase inATCase activity in
adenovirus-infected cells wasdemonstrated. To investigate the
mechanism ofcontrol of enzyme activity in HeLa cells, ATCasewas
partially purified (Consigli and Ginsberg,1964), and the effect of
end products as inhibitorswas determined in vitro. The results of a
repre-sentative experiment (Table 2) indicate that thecytidine
phosphates were effective enzymeinhibitors, and cytidine
triphosphate (CTP) wasa better inhibitor than either cytidine
diphos-phate (CDP) or cytidine monophosphate (CMP).Enzyme extracted
from infected cells was in-hibited to a greater extent than was
enzyme fromuninfected cells. Uridine monophosphate (UMP)and
thymidine monophosphate (TMP) did notreduce enzyme activity. The
evidence presentedimplies that ATCase in HeLa cells could be
con-trolled by feedback inhibition, that enzyme frominfected cells
is in a form more easily inhibitedthan that from uninfected cells,
and that cytidinephosphates (particularly CTP) are the
naturalfeedback inhibitors. These data bear a strikingsimilarity to
those reported by Gerhart andPardee (1962) for ATCase from E.
coli.
TABLE 2. In vitro inhibition of aspartate transcar-bamylase
activity by cytidine phosphates*
Per cent inhibitionCompoundoUninfected Infected
Cytidine triphosphate ...... 13.0 43.0Cytidine
diphosphate....... 12.0 36.0
Cytidine monophosphate .. 6.0 26.0Uridine monophosphate..... 0.0
5.0
Thymidine monophosphate . 0.0 0.0
* Monolayers were harvested 18 hr after infec-tion with a
multiplicity of 8 to 10 IDso . HeLa cellsfrom ten 32-oz bottles
were pooled, and ATCasewas purified by DEAE-cellulose
chromatography.The reaction mixture contained 1.5 X 10-2 M
car-bamyl phosphate (Li salt), 5.0 X 10-3 M aspartate,10-3 M
pyrimidine riboside phosphates (neutral-ized), 0.05 M tris-acetate
buffer (pH 8.5), and 0.27mg of protein (infected) and 0.23 mg of
protein(uninfected) per ml in preparations of partiallypurified
enzymes. Incubated at 35 C for 30 min.
TABLE 3. Effect of heat on the aspartate transcar-bamylase from
uninfected and type 5
adenovirus-inJected HeLa cells*
Uninfected cells Infected cellsTime heated Increase Incr
at 60 C Specific in Specific Ineaseactivityt activity activityt
activity
min % %0 0.20 0.581 0.28 40 0.56 -2 0.34 70 0.60 3.33 0.44 120
0.60 3.34 0.36 80 0.57 -5 0.26 30 0.52
* Monolayers harvested 18 hr after infectionwith 8 to 10 ID5s
per cell. To prepare enzyme, anequal number of cells were disrupted
and the cell-free lysates were heated for the indicated times inthe
presence of 2.0 X 10-4 M EDTA; 2.0 X 10-3 Mmercaptoethanol; and 5.0
X 10-3 M phosphatebuffer (pH 7.0). The enzyme was
immediatelychilled after heat treatment and assayed for ac-tivity.
The reaction mixture contained 7.5 X 10-3M carbamyl phosphate (Li
salt), 5.0 X 10-3 iuaspartate (Na salt), 0.05 M tris-acetate buffer
(pH8.5), and cell-free lysate from 2 X 106 cells. In-cubated at 35
C for 30 min.
t Specific activity = micromoles of ureidosuc-cinate synthesized
per 2 X 106 cells.
Effect of heat on activity of A TCase from unin-fected and type
5 adenovirus-infected HeLa cells.The results obtained suggested
that ATCase fromuninfected cells was in an inhibited state.
Bvanalogy with the characteristics of this enzymein E. coli
(Gerhart and Pardee, 1962), it waspostulated that ATCase in
mammalian cells mayhave a reactive site for feedback control
whichwas occupied in the uninfected cells, effecting aninhibited
state. To test this hypothesis, enzymesfrom uninfected and
adenovirus-infected HeLacells were heated at 60 C for 1 to 5 min,
andenzyme activity was measured. The data from arepresentative
experiment summarized in Table3 indicate that the activity of
enzyme fromuninfected cells increased gradually and reacheda
maximum after heating for 3 min. In contrast,after enzyme from
virus-infected cells wassimilarly heated, no significant increase
in en-zyme activity occurred.These data imply that (i) ATCase in
uninfected
HeLa cells is partially inhibited; and (ii) virusinfection, by
demanding increased nucleic acidbiosynthesis, removes the inhibitor
of ATCase
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CONSIGLI AND GINSBERG
I PsI Is
lII
1 VIe¢crsa-6/A603MiruuS
6.0 7.0 8o gqo
FIG. 1. Optimal pH levels for aspartate trans-carbamylase from
type 5 adenovirus-infected HeLacells before and after heating. The
enzyme reactionmixtures were prepared as described in Table
3.Tris-acetate buffer (0.05 M) at indicated pH levelswas used to
adjust the pH of the enzyme.
and consequently increases enzyme activity. It isstriking that
the activity of enzyme from in-fected cells could be inhibited
approximatelytwofold by CTP (Table 2) and, conversely,enzyme from
uninfected cells could be activateda similar degree by heat (Table
3).
Effect of heat on characteristics of A TCase fromuninfected and
type 5 adenovirus-infected HeLacells. ATCase from infected cells
was reported tohave a pH optimum and kinetics significantly
dif-ferent from enzyme extracted from uninfectedHeLa cells
(Consigli and Ginsberg, 1964). Thefinding that the activity of
ATCase from unin-fected cells could be increased by heat
suggestedthat this treatment might alter the pH optimumand the
kinetics of this enzyme so that it would besimilar to ATCase from
virus-infected cells. Thepostulate that enzyme from the infected
cells hada free inhibitor-reactive site would predict thatheating
of this enzyme would not alter its proper-ties. Enzyme activity
from uninfected and type 5adenovirus-infected cells was assayed
after heat-ing for 3 min at 60 C or after CTP treatment,and the
following properties were determined:pH optimum, Km, and maximal
velocity.
Optimal pH. The results of enzyme assays de-termined at pH 6.0
to 9.0 are summarized inFig. 1. The marked differences between the
un-heated enzymes from uninfected and infectedcells are clear. The
change effected by heatingATCase from uninfected cells is striking:
the pHoptimum was shifted from pH 7.5 to 8.5. Enzymefrom infected
cells had a pH optimum of 8.5,
which was unaltered by heat; the smal plateauat pH 7.0 to 7.5
did disappear, suggesting thatthe preparation from infected cells
containedsome enzyme in the inhibited state similar tothat in
control cells.Enzyme kinetics. ATCase from uninfected and
adenovirus-infected cells was partially purifiedby
chromatography on diethylaminoethyl(DEAE) cellulose, and the Km
values for asparticacid were determined by Lineweaver-Burke(1934)
plots. The results of a representativeexperiment (Table 4) indicate
again the distinctdifferences between enzymes from uninfected
andinfected cells. ATCase from infected cells had amarkedly
increased K,. for aspartate, 2.0 X 10-2M, and a maximal velocity of
1.62, as comparedwith enzyme from uninfected cells, 8.2 X 10-3M and
0.91, respectively. However, when enzymefrom control cells was
heated, the Km and maxi-mal velocity approached those of the
ATCasefrom infected cells. To determine further the rela-tionship
between enzymes from uninfected andinfected cells, CTP was added to
a preparationof each, and the K,, and maximal velocity
weredetermined. The kinetics of enzyme from in-
TABLE 4. Kinetics for aspartate transcarbamylasefrom uninfected
and type 5 adenovirus-
infected HeLa cells*
Maximal velocity(pnoles of ureido-
Treatment succinate syntle- Km (aspartate)sized per mg of
protein)
M
Uninfected ........... 0.91 8.2 X 10-3Infected..............
1.62 2 MX 10-2Uninfected (heated 60C,3 min) .......... 1.89 4.0 X
10-2
Infected (CTP, 2 X10-3M .............. 0.62 8.2 X 10 3
* Monolayers were harvested 18 hr after infec-tion with 8 to 10
IDs, per cell. HeLa cells from ten32-oz bottles were pooled, and
ATCase was purifiedby DEAE-cellulose chromatography. The
reactionmixture contained 1.5 X 10-2 M carbamyl phos-phate (Li
salt); 5.0 X 10-3, 1.0 X 10-2, 2.0 X 10-2,3.0 X 10-2, or 5.0 X 10-2
M asparate; 0.05 M tris-acetate buffer (pH 8.5); 2.0 X 10-3 M CTP
whenused; and 0.35 mg of enzyme protein (uninfected)and 0.40 mg of
enzyme protein (infected) per ml.Incubated 20 min at 35 C. Heat
treatment of theenzymes was performed as in Table 3. The Km andVmax
for aspartate were calculated for a 20-minreaction time.
.6]
to .5--JI
L .4-x9ceN,, .52'C).2
(A
NseecrED
I _ UNIAFCCTED
6.0 %0 8.0 4.O
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CONTROL OF ASPARTATE TRANSCARBAMYLASE
fected cells was similar to ATCase from controlcells when CTP
was present.
Purine and pyrimidine pools in uninfected andtype 5
adenovirus-infected HeLa cells. Yates andPardee (1957) demonstrated
that the activity ofbacterial ATCase was easily altered and
depend-ent on the intracellular concentration of metabo-lites,
which were not substrates of the enzyme butrather end products of
the metabolic pathway.Experiments were carried out to
determinewhether the increase in ATCase activity in virus-infected
cells was accompanied by a decrease inthe pyrimidines of the
cellular acid-soluble pool.Previous experiments (Table 1) had shown
thataddition of pyrimidines to the tissue cultureminimal medium
prevented increased ATCaseactivity in infected cells. HeLa cell
cultures in-fected for 18 hr (period of optimal ATCase activ-ity)
with type 5 adenovirus were employed. Table5 presents the findings
that were obtained bytwo-dimensional chromatography of the
acid-soluble pools in five experiments. The pyrimidineswere
decreased by 30 to 37%. Thymine, asmeasured by chromatography or by
microbio-logical assay, was not detectable. These resultsconfirm
data reported previously by Salzman(unpublished data). Uridine, as
identified bychromatographic and spectral analysis, wasconsistently
found in the acid-hydrolyzed ma-terial; all other nucleosides and
nucleotides werecompletely hydrolyzed to the respective bases.
DISCUSSION
Adenovirus infection of mammalian cells invitro initiated the
sequential biosynthesis ofRNA (9 to 14 hr), DNA (10.5 to 20 hr),
and virusantigens (14 to 20 hr), which was culminated bythe
assembly of infectious virus particles (Flana-gan and Ginsberg,
1962, J. Bacteriol. in press;Wilcox and Ginsberg, 1963). Shortly
afterinitiation of ribonucleic acid (RNA) synthesisand accompanying
DNA production, an in-creased activity of ATCase was detected
(Con-sigli and Ginsberg, 1964). ATCase from infectedcells had a pH
optimum, maximal velocity, andKm for aspartate different from the
enzyme fromcontrol cells. Although these data suggested thatATCase
from infected cells was an isozyme, theevidence described in this
communication clearlyindicated that this was not the case. The
resultsreported indicate that, with increased nucleicacid
biosynthesis in infected cells, the feedback
TABLE 5. Comparison of the acid-soluble pool ofuninfected and
type 5 adenovirus-infected
HeLa cells*
CompoundAmt (miAmoles/106 cells) Per centUninfected Infected
depleted
Adenine ...... 2.36 1.81 24Guanine ...... 0.54 0.39 28Cytosine .
... 0.20 0.13 34Uracil ........ 0.74 0.48 37Uridine ....... 0.95
0.70 26
* Monolayers were harvested 18 hr after infec-tion with a virus
multiplicity of 8 to 10 ID 50Data represent the geometric means of
resultsfrom five experiments.
inhibitor, CTP, is released from the feedbacksite on the enzyme
molecule; that release of feed-back inhibition results in increased
enzymeactivity, and that enzyme whose inhibitor site isunoccupied
has characteristics identical to thosedescribed for ATCase in
adenovirus-infected cells.Thus, enzyme from uninfected cells could
beconverted by heat (60 C for 3 min) to the reac-tivity of ATCase
from infected cells. Conversely,enzyme from infected cells could be
transformedby addition of CTP so that it assumed theproperties of
ATCase from uninfected cells. In-deed, preliminary experiments
indicate that 24 hrafter infection of HeLa cells with type 5
adeno-virus, ATCase has the same Vmax and Km withaspartate as does
enzyme from uninfected cells(unpublished data).Accompanying
increased nucleic acid synthe-
sis, the concentration of pyrimidines and purineswas decreased
in the total acid-soluble pool.Pyrimidines showed the greatest
depletion, about35%, and it may be considered that the depletionof
pyrimidines resulted in release of feedbackinhibition. It may
appear puzzling that a decreasein concentration of only 30 to 35%
should effecta marked release of feedback control; however,it
should be considered that analyses were madeof the total pyrimidine
pool which was measuredas bases. Thus, the cytidine pool was
derivedfrom the base, the ribo- and deoxyribosides, andthe mono-,
di-, and triphosphate nucleosides. Thedata obtained indicate that
the phosphorylatedderivatives served as inhibitors and that CTPwas
most effective. Bresnick (1962) recently re-ported that
deoxyribonucleotides as well asribonucleotides can act as feedback
inhibitors ofATCase in rat liver.
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CONSIGLI AND GINSBERG
Ennis and Lubin (1963) reported that ATCaseis regulated by
end-product repression in sar-coma 180 cells propagated in vitro.
Experimentscarried out during the course of the
presentinvestigation demonstrated that thymidine orcytidine
inhibited the increase in ATCase whenadded to infected cultures up
to 8 to 10 hr afterinfection. Thereafter, the pyrimidine
nucleosidehad a decreasing inhibitory effect (unpublisheddata). The
results could be interpreted to indi-cate a repression of enzyme
biosynthesis bythymidine or cytidine during early stages of
themultiplication cycle. In the face of clear-cutevidence for
feedback inhibition and withoutdirect evidence for synthesis of new
enzymeprotein, the conclusion that ATCase is regulatedby
end-product repression as well as feedback in-hibition seems
hazardous.
It is striking that the control mechanism forATCase in HeLa
cells appears similar to, if notidentical with, ATCase from E. coli
(Gerhart andPardee, 1962); the enzyme has an active site
forfeedback inhibition and active substrate sites foraspartic acid
and carbamyl phosphate. However,distinct differences between ATCase
from E. coliand HeLa cells are apparent: (i) CTP has agreater
inhibitory effect on the bacterial enzyme(85 to 86%; Gerhart and
Pardee, 1962) than onATCase from adenovirus-infected cells (43
to50%); (ii) ATCase from control E. coli can be in-hibited by CTP
(cytosine derivatives), butenzyme from uninfected HeLa cells is
unaffectedby cytosine derifatives; and (iii) the kinetics
ofunheated or heated enzyme from uninfected oradenovirus-infected
HeLa cells are similarand simple, and are unaffected by heat
(Con-sigli and Ginsberg, 1964). In contrast, ATCasefrom E. coli has
complex kinetics which aredifferent from the kinetics of enzyme
which washeated or chemically treated (Gerhart andPardee, 1962).
The regulation of enzyme ac-tivity during virus multiplication
responds todemands for increased nucleic acid biosynthesisand is
controlled by mechanisms which areeffective during physiological
demands in bac-terial and probably mammalian cells.
ACKNOWLEDGMENTS
This research was supported in part by agrant from the National
Institute of Allergy andInfectious Diseases, U.S. Public Health
Service.The advice on enzymological matters given by
Lewis Pizer, Department of Microbiology,University of
Pennsylvania, throughout theseinvestigations was invaluable.
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