Download Feedback Inhibition ofAspartate

Feedback Inhibition of Aspartate Transcarbamylase
in Liver and in Hepatoma*
EDWARDBRESNICK
(Department of Biochemistry, Baylor University Collegeof Medicine, Houston, Texas)
SUMMARY
Aspartate transcarbamylase was partially purified from rat liver, Morris hepatoma
5123-A or D, Hepatoma 7800, and Novikoff hepatoma and was shown to be inhibited
by pyrimidine deoxyribonucleosides and deoxyribonucleotides. The pyrimidine ana
logs, 5-bromo-2'-deoxyuridine, 5-bromouridine, 5-bromo-2'-deoxycytidine,
5-fluoro2'-deoxyuridine, 5-fluorouridine, 5-fluoro-2'-deoxycytidine, 5-iodo-2'-deoxyuridine, al
so inhibited enzymatic activity. The enzyme, however, was most effectively in
hibited by 2'-deoxyadenosine-5'-monophosphate
(or 2'-deoxyguanosine-5'-monophosphate [d-DMP]) and by 2'-deoxyadenosine or 2'-deoxyguanosine.
Aspartate transcarbamylase was shown to be identical in liver and in the hepatomas
by pH optimum, Km values for carbamyl phosphate and aspartate, Vmax,degree of
inhibition by Cu44", by the pyrimidines, and by the inhibition constants for BCDR,
FUDR, thymidine, and d-AMP (or d-GMP).
It was concluded that negative feedback inhibition of pyrimidine biosynthesis oc
curs in hepatomas as well as in liver, and to the same extent. Furthermore, the control
of aspartate transcarbamylase by d-AMP (or d-GMP) may play a significant role in
VIVO.
The original deletion hypothesis for carcinogenesis proposed by Potter (17) in 1944, which
stated that neoplasia may be characterized by an
absence or reduction in concentration of one or
more enzymes, has been modified recently (18) to
include the mechanisms responsible for the control
of enzyme activity or synthesis. These mechanisms
include negative feedback inhibition operative at
the substrate level and repression operative at the
genome. Aspartate transcarbamylase, an enzyme
responsible for the irreversible carbamylation of
L-aspartic acid by carbamyl phosphate (I) is sub
ject to both negative feedback inhibition (24, 27)
and to repression (28) in Escherichia coli.
COOH
Negative feedback inhibition by pyrimidines of
this enzyme is also manifested in mammalian tis
sues—i.e., in Ehrlich ascites cells (4) and in liver
preparations (2). No repression of this enzyme has
been observed in liver by pyrimidines or pyrimi
dine derivatives to date.1
In the absence of repression in mammalian tis
sues, the role of feedback inhibition of pyrimidine
biosynthesis as a mechanism of enzymatic control
would assume added importance. Therefore, an in
vestigation has been undertaken to ascertain the
relative importance of such regulatory control in
the development of normal liver and of hepatomas.
Toward this end, aspartate transcarbamylase has
COOH
O
O
CHNH2
PO3H2->CH—NH—C—NH2
:HNH2 +
+ NHj—C—O—
NH
H3PO4
(I)
CH2
CH2
COOH
COOH
* This investigation was supported by an American Cancer
Society Institutional Research grant and by a grant from the
Anna Fuller Fund.
been partially purified from rat liver and from vari
ous rat hepatomas, and the effects of various pyrim
idine derivatives upon this enzyme have been
' E. Bresnick, unpublished experiments.
Received for publication July 11, 1962.
1246
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BRESNICK—Asportate Transcarbamylase
investigated. The results of these studies are re
ported in this manuscript.
MATERIALS AND METHODS
Chemicals.—Allpyrimidine and purine nucleotides were obtained from commercial sources. The
author is indebted to the Hoffmann-LaRoche Co.,
Nutley, N.J., for FUDR,2 FUR, and FCDR and
to Dr. Samuel Bieber, Burroughs Wellcome & Co.,
Inc., Tuckahoe, N.Y., for gifts of BCDR, BUDR,
and IUDR. Commercial dilithium carbamyl phos
phate (90 per cent pure) was further purified by
the method suggested by Gerhart and Pardee (5)
and stored over calcium chloride in a desiccator at
-5°C.
Animals.—Livers from male Holtzman rats
weighing from 125 to 150 gm. were employed as the
source of the enzyme. The author is also grateful
to Dr. Harold P. Morris of the National Cancer
Institute, Bethesda, Md., who provided the Morris
Hepatoma 5123, Hepatoma 7800, Hepatoma H-35,
and the Hepatoma 7316-A, as well as the informa
tion about these hepatomas. Buffalo rats bearing
the Morris Hepatoma 5123-A which had been in
oculated subcutaneously and bilaterally were sacri
ficed when the tumor was 54 days old. A Buffalo
rat bearing the Hepatoma 5123-D inoculated in
tramuscularly and subcutaneously on the right
side was sacrificed when the tumor was 87 days
old. The Hepatoma 7800 was obtained from a Buf
falo rat given inoculations intramuscularly and
subcutaneously and was used when 100 days old.
The Hepatoma 7800 is a well differentiated hepatocellular carcinoma induced in a male rat by N(2-fluorenyl)phthalamic acid (13), the same com
pound used to induce the Morris Hepatoma 5123.
Additionally, aspartate transcarbamylase activity
was determined in a 169-day-old Hepatoma 7316-A
obtained from a Buffalo rat which had been given
inoculations bilaterally and intramuscularly, and
in the Hepatoma H-35, obtained from the A X C
rat previously given inoculations intramuscularly
and subcutaneously. The Hepatoma 7316-A is a
slowly growing tumor induced originally by 2,4,6trimethylaniline in the Buffalo rat, whereas the
Hepatoma H-35 was originally induced by Reuber
(23) in the gray A X C rat. The initial Novikoff
2The following abbreviations are used: BUDR, S-bromo-2'deoxyuridine; FUDR, 5-fluoro-2'-deoxyuridine; FUR, 5-fluorouridine; FCDR, 5-fluoro-a'-deoxycytidine; BCDR, 5-bromo2'-deoxycytidine; IUDR, 5-iodo-2'-deoxyuridine;d-UR, deoxyuridine; d-UMP, 2'-deoxyuridine-5'-monophosphate; UMP, uridine-5'-monophosphate; d-CR, 2'-deoxycytidine; d-CMP, 2'deoxycytidine-5'-monophosphate; CMP, cytidine-5-monophosphate; TdR, thymidine; TMP, thymidine-5'-monophosphate;
d-AMP, 2'-deoxyadenosine-5'-monophosphate; d-GMP, 2'-deoxyguanosine-5'-monophosphate; d-AdR, 2'-deoxyadenosine;
d-GuR, 2'-deoxyguanosine; BUR, 5-bromouridine.
in Liver
1247
hepatoma was kindly supplied by Dr. N. B. Fur
long of the M. D. Anderson Hospital and Tumor
Institute, Houston, Texas. The tumor was main
tained by weekly transfers, intraperitoneally, into
Holtzman rats. The ascitic hepatoma cells were
collected by centrifugation, washed twice with 0.9
per cent saline, weighed, and resuspended to a con
centration of 10 per cent with distilled water.
Enzyme preparation.—Homogenates of 10 per
cent in 0.25 Msucrose were prepared from the liver
and hepatomas (except the Novikoff hepatoma,
which was suspended in water) and were centrifuged at 105,000 X g for 45 minutes at 4°C. All
the steps in the purification procedure were con
ducted at this temperature. The purification pro
cedure has been described in detail elsewhere (3)
and will only be presented briefly here.
To the supernatant preparation obtained after
centrifugation was added sufficient (NHOzSC^ to
give a 30 per cent saturation. The resultant pre
cipitate was dialyzed against 0.01 M imidazole
buffer, pH 7.0, containing 0.002 M mercaptoethanol, for 1 hour in a rotating dialyzer apparatus,
then calcium phosphate gel (6) was added (2 mg/
mg protein). The enzyme was eluted from the gel
with 0.3 Mphosphate buffer, pH 7.4, and was then
dialyzed against the imidazole-mercaptoethanol
buffer for 1 hour in the dialyzer. The enzyme was
further fractionated on DEAE-cellulose columns
(1.5 X 30 cm.) by discontinuous elution with the
imidazole-mercaptoethanol buffer, containing 00.3 Mpotassium chloride. The total enzymatic ac
tivity was eluted withJO.3 MKC1. This preparation
could be maintained for 5 days at 4°C. without
loss in activity but could not be stored below freez
ing.
Protein was assayed by the colorimetrie proce
dure of Lowry et al. (IO) or the spectrophotometric
method based upon the absorbance at 280 mju (8).
Enzyme assay.—Enzymatic activity was as
sayed by the production of carbamylaspartate in
a system containing the following: carbamyl phos
phate, 10 Amóles;L-aspartic acid, 3.75 Amóles;en
zyme; pyrimidines or purines; 0.2 M Tris buffer,
pH 9.2, to make a total volume of 1.5 ml. The
incubation was conducted at 37°C. for 30 minutes,
after which enzymatic activity was stopped by the
addition of 0.5 ml. of 4 N perchloric acid. The car
bamylaspartate concentration was determined in
a 0.5-ml. aliquot by the recent modification (5) of
the method of Koritz and Cohen (7). The amount
of carbamylaspartate produced nonenzymatically
was simultaneously determined.
RESULTS
A summary of the scheme for the purification
of aspartate transcarbamylase from liver, the No-
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1248
Cancer Research
vikoff hepatoma, Hepatomas
5123-A or D, and
7800 is presented in Table 1. An over-all purifica
tion of 56-fold was achieved from rat liver, whereas
the purification from the three hepatomas varied
from 30- to 54-fold. Only the Novikoff hepatoma
was significantly different from liver when the aspartate transcarbamylase
activity was expressed
on a wet weight basis. The values for liver, Morris
Hepatoma 5123-A, 5123-D, Hepatoma 7800, Hepa
toma H-35, Hepatoma 7316-A, and Novikoff hepa
toma were 20.0, 21.1, 26.4, 27.8, 34.8, 20.1, and
96.9 units per gram wet weight, respectively.
TABLE 1
PARTIALPURIFICATION
OFASPARTATE
TRANSCAR
BAMYLASE
FROMLIVERANDHEPATOMAS
Vol. 22, November
1962
koff and 5123-D hepatomas by the purine deriva
tives was also observed.
Pyrimidine analogs proved to be effective in
hibitors of the partially purified enzyme of liver
as well as of the hepatomas (Table 3). The deoxy
ribonucleosides of bromouracil and fluorouracil
were slightly more active as inhibitors than the
corresponding ribonucleoside. The most potent in
hibitor of the pyrimidine analogs was BCDR,
which at 50 /¿moles
resulted in a reduction of liver
aspartate transcarbamylase by 69.6 per cent. Once
again, the pyrimidine analogs exerted comparable
inhibitions upon the enzymes isolated from the
Novikoff or 5123-D hepatomas.
In Chart 1 the inhibitions of the liver aspartate
TABLE2
512S-A*HEPATOMA
7800NOVIKOFF
HEPATOMA(Units/mg
t0.040
protein)
034
10% Homogenate
106Xy Superna te
0.090
0.100
0.14
1.07
0.59
0.85
0.26
1.86
(NH4)2SO4,80%
1.242.28560.030
0.701.39430 0.901.84540.36
4.9110.630
eluateDEAE
Gel
fractionPurification,X-foldLIVERHEPATOMA
INHIBITION OF ASPARTATETRANSCARBAMYLASE
FROM
LIVERANDHEPATOMAS
BYNUCLEOSIDES
ANDNUCLEOTIDES
ENZYMELiver31.554.711.520.041.320.041.150.121.643.010.020.044.3
OF
ibition)47.117.117.246.227.27.434.135.768.279.47
(%W49.917.436.123.049.636.115.148.622.252.551Z
d-URd-UMPUMPd-CRd-CMPCMPTdRTMPd-AMP
* Similar purification was obtained with the Morris hepato
ma 5123-D.
t One unit is defined as the number of /¿molesOf product
(carbamylaspartate) produced under the standard conditions
of the assay.
The effect of pyrimidine and purine nucleosides
and nucleotides upon the partially purified aspartate transcarbamylase from liver and from the
hepatomas was determined (Table 2). The deoxyribonucleosides were very effective inhibitors of
this enzyme from all sources investigated, and thymidine appeared to possess the greatest inhibitory
efficacy of the pyrimidine derivatives; the deoxyribonucleoside of uracil was as effective as the cor
responding cytosine compounds. The nucleotides,
however, of uracil, cytosine, or thymine were not
as inhibitory as the nucleosides. The degree of
(or
inhibition of the enzyme from the hepatomas was d-GMP)d-AdR
11.0
52020 30.065.061.2
similar to that observed with the liver enzyme.
The deoxyribonucleosides and deoxyribonucleo(or
tides of adenine and guanine were much more ef
d-GuR)MMOLE8205010205020205020502010205010205012
50SOURCE
83.0Novikoff
fective than thymidine (by approximately four
fold), and the inhibition produced by the nucleoside
The results are reported as an average of two experiments.
was equal to that observed with the nucleotide.
The amounts of carbamylaspartate produced by the enzyme
from the livers, Novikoff hepatoma, Hepatoma 5123-D, and
The corresponding ribose derivative, at compara
Hepatoma 7800 in Experiment 1 was 0.415, 0.427, 0.375, and
ble concentrations, exerted no inhibition upon 0.398
/¿moles;
in Experiment 2,0.397, 0.445, 0.397, and 0.356
this enzyme. Comparable inhibition of the Novi
limóles.
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BRESNICK—Asportate Transcarbamyla.se
transcarbamylase by TdR, FUDR, BCDR, d-AMP
(or d-GMP) have been plotted according to the
method of Lineweaver and Burk (9). The data
clearly define the inhibition by these compounds
as competitive. The inhibition constants are pre
sented in Table 4 and will be discussed subse
quently. Similar Lineweaver and Burk plots have
been obtained with the hepatoma enzymes.
A comparison of the aspartate transcarbamylase
enzymes partially purified from liver, Morris Hepa
toma 5123-A or D, Novikoff hepatoma, and Hepa
toma 7800 is presented in Table 4. The pH opti
mum of the enzymes was 9.2; the Km for aspartate
did not vary significantly and ranged from 1.6 to
2.2 X 10~3M; the Km with carbamyl phosphate
in Liver
1249
inhibition of the enzymes isolated from liver and
from the various hepatomas revealed no essential
difference. Furthermore, the inhibition constants
for FUDR, BCDR, TdR, d-AMP (or d-GMP) did
not significantly differ in hepatomas from that ob
served with liver. It is interesting to note that
d-AMP (or d-GMP) was the most potent of the
inhibitors in this series and was bound to the en
zyme approximately 10 times better than was aspartic acid. The above data appear to indicate
TABLE 3
INHIBITIONOF ASPARTATE
TRANSCARBAMYLASE
FROM
LIVERANDHEPATOMAS
BYPYRIMIDINE
ANALOGS
ENZYMELiverNovikoff5ÌÌS-D(%
OF
Inhibition)29.0
BUDRBURFUDRFURIUDRBCDRFCDRMMOLE810205020
86.2
59.427.5
5010
42.128.0
20
5020
36.2
61.636.3
5020
42.117.0
5010
46.629.0
20
53.9
5020SOURCE
69.624.746.3
61.219.2
I/S
(xlCT)
41.519.1
335.641.219.6
36
CHART1.—Inhibition of aspartate transcarbamylase (liver)
by pyrimidine nucleosides. The liver enzyme was incubated
with carbamyl phosphate, 10 Amóles;L-aspartate, as indicated
the chart; 0.2 MTris buffer, pH 9.2; alone (bottom line) or
40.439.656.8in
with either BCDR, 6.7 X 10~3M; FUDR, 6.7 X IQ-'M; dAMP, 3.3 X 10-»
M; or TdR, 6.7 X 10~3M.
53.835.427.620.7
The results are the average of two experiments. The
amounts of carbamylaspartate produced by the enzyme from
the liver, Novikoff hepatoma, and hepatoma 5123-D were
0.275, 0.250, and 0.296 /jmole (in Experiment 1); in Experi
ment 2, 0.395, 0.306, and 0.321 jumóle.
as substrate also did not vary significantly and
ranged from 3.0 to 4.8 X 10~4M; the Vmaxalso did
not differ and ranged from 1.6 to 2.5 X 10~4M.
Liver aspartate transcarbamylase was inhibited by
heavy metals—i.e., GU++, Hg++, Cd"1"*",
and Ag+
(3) ; this was relieved upon the addition of EDTA
to the medium. An examination of the relative
that aspartate transcarbamylase is the same in the
liver and in the hepatomas, independent of the
growth rate of the hepatoma.
DISCUSSION
The group of the slowly growing hepatomas em
ployed in this study, Hepatomas 5123-A or D.7316A, 7800 or H-35, possess aspartate transcarbamyl
ase activities equivalent to that observed in adult
normal liver. Previously, it had been noted that
the Morris Hepatoma 5123 possessed primarily an
aerobic energy metabolism, resembling adult liver
(1); a carbohydrate enzyme composition close to
liver (25), although there did occur a decreased
glycogen synthesis from labeled carbon and a de
pressed phosphoglucomutase (26); a similar com
position of enzymes involved in the anabolism and
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1250
Cancer Research
catabolism of pyrimidines (11, 20). The Novikoff
hepatoma, on the other hand, was at the other
end of the spectrum. A comparative enzymology
of the Novikoff hepatoma and liver indicated dif
ferences in the enzymes involved in carbohydrate
metabolism (16, 26), in pyrimidine anabolism and
catabolism (11, 12, 19, 20), as well as other en
zymes (15). The activity of aspartate transcarbamylase may also be employed to illustrate this
pattern—e.g., the Morris Hepatoma 5123 more
closely resembles liver. It is also interesting that
the activity of the next enzyme involved in the bio
synthesis of pyrimidines, dihydroorotase, is identi
cal in the Morris Hepatoma 5123 and in the liver.1
The results presented in this report agree essen
tially with those obtained with crude liver prepa
rations (2) and with Ehrlich ascites cells (4) but
do not conform to the results observed in bacterial
systems (5, 27). However, the different require-
Vol. 22, November
1962
trol of both the de novo synthesis of pyrimidines
and the reduction to deoxyribonucleotides by the
deoxyribonucleotides of thymine and adenine (or
guanine)—may play a significant role in the mam
malian cell.
The data presented indicate that aspartate
transcarbamylase, the locus for negative feedback
inhibition in E. coli (24, 27), Ehrlich ascites cells
(4), and in liver (2), is not significantly different
in either slowly growing hepatomas, a fast-growing
hepatoma, or normal liver. This conclusion is based
upon the similarity of Km values for aspartate,
carbamyl phosphate, the Vmax,the pH optimum,
inhibition by Cu++, and the inhibition constants
for FÃœDR,BCDR, TdR, and d-AMP (or d-GMP).
Negative feedback inhibition of aspartate trans
carbamylase, therefore, appears to operate equally
well in hepatomas or in liver in vitro.
This regulatory mechanism, if operative in vivo,
TABLE4
COMPARISON
OFTHEPROPERTIES
OFASPARTATE
TRANSCARBAMYLASE
FROMLIVERANDHEPATOMA
S1ÕS-A*9.22.2X10-'
optimumK„
pH
aspartateKm
—
phosphatev„„Inhibition
—carbamyl
M)K¡: by CuSO4 (S.SXKT4
FUDRBCDRTdRd-AMP
(or d-GMP)Liver9.21.9X10-'
M4.8X10-'M1.9X10-'
M8.0X10-*
M2.5X10-'
M65.0%S.
M2.0X10-'
M59.4%4.3X10-«M2.
M61.3%1.6X10-'
M2.6X10-'
4X10-'
M1.5X10~3M1.6X10-'
M4.
M1.6X10-'
1X10-'
M60.6%
M2.6X10-*
M2.6X10-'M78009.21.6X10-*
OXIO-3 MNovikoff9.22.0X10-»M3.6X10-*
MMorris
* Similar results were obtained with the Morris Hepatoma 5123-D.
ments for feedback inhibition for the two systems
are not surprising, since the two enzymes do differ
markedly in properties (3, 5).
Gerhart and Pardee (5) initially observed the
inhibition of E. coli aspartate transcarbamylase
by guanosine triphosphate but found that the lat
ter was not as effective as deoxycytidine triphos
phate. In the partially purified liver system, dAMP (or d-GMP) was much more effective than
the pyrimidine. Regulatory control of pyrimidine
biosynthesis by purine derivatives has already
been recorded. Reichard and co-workers (21, 22)
observed the inhibition of the reduction of cytidine
diphosphate to the corresponding deoxy- deriva
tive by deoxyguanosine triphosphate and deoxyadenosine triphosphate and, to a lesser extent, by
thymidine triphosphate. A similar observation has
been made by Morris and Fischer (14), who re
ported the inhibitory action of thymidine upon
the conversion of cytidylic acid to d-CMP in tissue
culture. This regulatory mechanism—e.g., the con-
does not appear to play any significant role in the
development of hepatoma. An alternative explana
tion, however, may lie in the amounts of pyrimi
dine or purine derivatives present within the hepa
toma cell.
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Feedback Inhibition of Aspartate Transcarbamylase in Liver and
in Hepatoma
Edward Bresnick
Cancer Res 1962;22:1246-1251.
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