[CANCER RESEARCH 39, 436-442, February 1979]
0008-5472/79/0039-0000$02.00
Demonstrationof Two Components and Association of Adenosine
Diphosphate-CytidineDiphosphate Reductase from Cultured
Human LymphoblastCells (Molt-4F)1
Chi-Hsiung Chang and Yung-chi Cheng2
Department of Experimental Therapeutics, Roswell Park Memorial Institute, New York State Department of Health, Buffalo, New York 14263
ABSTRACT
Under the influence
Ribonucleotide meductase was isolated from a human
lymphoblast line (Molt-4F). Most of the meductase activity
was present in the cytosol fraction. Two components (A and
B) were found which were readily separable by deoxy
guanosine triphosphate Sepharose column chromatogma
phy. Only Component B was retained on this column and
could be eluted by high concentrations of KCI. Components
A and B were purified further by blue Sepharose, diethyl
aminoethyl cellulose and phenyl-Sephamose column chro
matography, as well as by sucrose gradient sedimentation.
The apparent molecular weights estimated by sucrose gra
dient sedimentation were 100,000 for both Components A
and B, and 210,000 for the nondissociated mibonucleotide
reductase. The cytidine diphosphate (COP) and adenosine
diphosphate (AOP) reductase activities cochnomatogmaphed
throughout
the purification
procedure
with a constant
ratio
of 1.73 ±0.19 (5.0.) Variation of the ratio of purified
Component A to B led to subsequent variation in overall
activity. However, the ratio of COP to AOP enzyme activity
remained constant. The enzyme activities of reconstituted
purified A and B components were further characterized
with
reference
to cation
requirements.
Of those divalent
cations tested, magnesium ion was found to be essential
for maximal enzyme activity, while calcium ion gave only
partial activation. Addition of zinc or manganese ion, at
concentrations higher than 0.4 mM, to the reaction mixture
containing 6 mM MgCl2 caused a marked inhibition of the
enzyme activity for both ADP and CDP reduction. Spermi
dine and spemmine can partially
replace the MgCI2 require
ment for COP and ADP reduction. The optimal concentra
tions of MgCl2 and dithiothreitol were 6 and 3 mM, mespec
tively.
Ribonucleotide reductase is the key enzyme responsible
for the synthesis
of deoxynibonucleotides
via the direct
reduction of mibonucleotides. The enzyme from Escherichia
coli has been purified
and well characterized
(2, 17, 27). It
is made of 2 nonidentical subunits, B1 and B2, both of
which are required to form the enzymatically active complex
in the presence of magnesium ion (4). The enzyme contains
nonheme iron which is essential for enzyme activity (3).
activators,
the same en
liven (19) have been reported.
More than one subunit
has
been shown for the enzyme derived from rabbit bone
marrow (18), matNovikoff hepatoma (23), and Ehnlich ascites
cells (12). The possible existence of different enzymes
responsible
for the reductions
of ADP and COP has been
proposed in the case of Chinese hamster cells (24), rat
regenerating liven (10), and Ehmlich ascites cells (13). No
detailed study of the isolation and properties of nibonucIe
otide reductase from human origin has been reported.
Because this enzyme has the potential of being a target for
cancer chemotherapy, we have undertaken the study of the
properties of the enzyme derived from a cultured human
lymphoblast
cell line (Molt-4F).
In this communication,
we
demonstrate that the enzyme consists of at least 2 compo
nents and that the ADP and COP meductase activities were
associated throughout the purification procedure. We also
describe some of the properties of the 2 components.
A
preliminary report of this work has appeared previously (5).
MATERIALSAND METHODS
The sodium salts of COP, ADP, ATP, and dGTP; DTT,3
HEPES, pymuvatekinase, lactic dehydrogenase, and hemo
globin
were all purchased
from Sigma Chemical
Co. , St.
Louis, Mo. Ammonium salts of all 14C-labeled nucleotides
were supplied by Amersham/Seamle Corp. , Arlington
Heights,
INTRODUCTION
of different
zyme molecule is capable of catalyzing the reduction of all
4 natural mibonucleotides at the diphosphate level (20).
The enzyme obtained from mammalian cells has not been
completely described due to difficulties in purification.
Studies of some properties of the partially purified enzyme
derived from matNovikoff hepatoma (22, 23), Ehnlich ascites
cells (13), rabbit bone marrow (18) and regenerating rat
Ill. Oowex 1-Cl was obtained
from Bio-Rad
Labo
natory, Richmond, Va. All materials required for cell cul
tunes were from Grand Island Biological Co. , Grand Island,
N. Y. All other chemicals were of reagent grade. dGTP
Sepharose was generously provided by Hoffmann and Blak
ley (16). Blue Sepharose,
Sephanose were purchased
cals, Piscataway, N. J.
DEAE-celiulose,
from Pharmacia
and phenyl
Fine Chemi
Culture Conditions. Molt-4F cells, which were isolated
from peripheral blood of acute lymphocytic
leukemia pa
tients, were cultured in 1-liter spinner flasks with Roswell
Park Memorial Institute Medium 1640 containing 5% heat
inactivated fetal calf serum, penicillin (100 units/mI), and
1 This
work
was
supported
by
USPHS
Project
Grant
CA-18499
and
Core
Grant CA-13038 from the National Cancer Institute.
2 An
American
Leukemia
Society
Scholar.
To
whom
should be addressed.
Received June 5, 1978: accepted November 3, 1978.
436
requests
for
stneptomycin
sulfate (100
@g/ml).The cells were maintained
reprints
3 The
abbreviations
used
are:
DTT,
dithiothreitol;
HEPES,
4-(2-hydroxy
ethyl)-1-piperazmneethanesultOnic acid.
CANCER RESEARCH VOL. 39
Downloaded from cancerres.aacrjournals.org on June 15, 2017. © 1979 American Association for Cancer Research.
Two Ribonuc!eotide Reductase Components from Lymphob!ast Ce!!s
with 25 strokes in a Dounce homogenizer. The homogenate
in the log phase of growth by feeding the cultures with an
was centrifuged at 100,000 x g for 60 mm, and the super
equal volume of the fresh medium every 24 hr. The cultured
cells were harvested by centnifugation and washed 6 times
natant (16 ml) was used immediately in the next purification
with phosphate-buffered saline (pH 7.2, 1 liter containing
step.
StreptomycinSulfate Fractionation.A solutionof strep
0.1 g CaCI2, 0.2 g KCI, 0.2 g KH2PO4,0.1 g MgCl2•6H20,
8g
NaCI, and 2.16 g Na2HPO4•7H20).
After this treatment, the
tomycin sulfate (20%, w/v) was added dropwise to the
cells were stored at —70°
until needed.
crude extract (16 ml) to yield a final concentration of 1%.
The solution was stirred for 20 mm at 4°,and the precipitate
Enzyme Assay. COP reductase was assayed by the
was removed by centnifugation at 10,000 x g for 20 mm.
method of Steepen and Steuart (25) with the use of Dowex
The supemnatant (16 ml) was used in the following step.
1-borate ion-exchange chromatography. The assay mixture
Ammonium Sulfate Fractionation. Ammonium sulfate
contained, in a final volume of 0.2 ml, [‘4CJCOP
(0.2 pCi;
0.15 mM), OTT (3 mM), MgCI2 (6 mM), ATP (5 mM), and a was added to the supemnatant obtained from the previous
step to 35% saturation. After a stirring at 4°for 30 mm, the
specified amount of the enzyme. AOP neductase activity
was determined by the method of Conyet a!. (14). The assay
suspension was centrifuged at 10,000 x g for 20 mm, and
mixture contained, in a final volume of 0.2 ml, [‘4CJADP the precipitate was discarded. More ammonium sulfate was
added to the supennatant to give 50% saturation. After
(0.22 pCi; 0.15 mM), OTT (3 mM), MgCI2 (6 mM), and dGTP
being stirred for another 30 mm, the precipitate was col
(5 mM), and a specified amount of the enzyme. An enzyme
sample heated for 2 mm in a boiling water bath prior to the
lected by centnifugation and was dissolved in 4 ml of Buffer
B. The enzyme solution was dialyzed overnight against the
addition of the labeled substrate served as the reaction
same buffer.
blank. The incubation was at 37°for 60 mm, and the
Separation of Components A and B on dGTP-Sepharose
reaction was linear with respect to time and enzyme con
centration during this incubation period. The inclusion of
Chromatography.The dialysate(4 ml) was madeto 10 mM
ATP in the COP meductase assay and dGTP in the ADP
with respect to NaF and loaded on a dGTP-Sephamose
meductase assay was essential for COP and AOP meductase column (1.5 x 10 cm) previously equilibrated with Buffer B
containing 10 mM NaF. The column was washed with the
activities, respectively. The specificity of the activators for
COP and ADP reductase activity will be reported in a same buffer until the absorbance at 280 nm was less than
0.05. Four consecutive-step elutions were then performed
subsequent communication. A preliminary report of the
with 0.5 mM dGTP, 50 mM KCI, 1 M KCI, and 2 M KCI in
kinetic behaviors of this enzyme has appeared previously
Buffer B as indicated in Chart 1. After dialysis against Buffer
(6). The activities of Components A and B as shown in
Charts 1 to 5 and Table 1 were determined by adding an
C, the fractions were analyzed for protein concentration
excess amount of B on A, respectively. The amount of
and enzyme activity. No activity was detected in any of the
Component A on B used to saturate the respective compo
fractions collected. Fractions 3 to 12, 13 to 19, 20 to 26, 27
nent under investigation was sufficient to give a minimum
to 31 , and 32 to 38 were pooled and dialyzed overnight
activity of 90 pmol COP reduced per mm pen ml and 50 pmol
against Buffer C containing 30% sucrose. Various combi
ADP reduced per mm per ml of Component A on B. These
nations of each pooled fraction were assayed for both AOP
components were obtained from the dGTP-Sephamose col
and COP reductase activities. Only the combination of the
umn.
pooled fractions from 3 to 12 (Component A) and 27 to 31
Cellular Fractionation.The proceduresusedfor obtain (Component B) gave both AOP and COP meductaseactivity.
ing various subcellulam fractions of the MoIt-4F cells have
The other pooled fractions could neither enhance non
inhibit the activity observed with this combination of Corn
been previously described (8).
Protein Determination. Proteinwas determinedby the
fluorometnic method of BOhlen and Stein (1). Bovine serum
albumin was used as the standard.
Enzyme Purification. All steps were performed at 0-4°as
indicated in Table 1 within a period of no more than 4 days.
The final preparation of the enzyme and aliquots of partially
purified components were stoned at —70°.
Under these
conditions, no significant loss of the enzyme activity occurs
during 1 week. Buffers used for the purification steps are as
follows. Buffer A contains 100 mM HEPES (pH 7.5), 1 mM
MgCl2, and 2 mM OTT. Buffer B includes 50 mM HEPES,
(pH 7.5), and 2 mM OTT. Buffer C contains 50 mM HEPES
(pH 7.5), 1 mM MgCI2, and 2 mM OTT, and 0.05 mM EOTA.
Details of the purification procedure are described under
“Results.―
RESULTS
Purification of Ribonucleotide Reductase
Preparationof Crude Extract. Molt-4Fcells (about9 x
10@cells) were suspended in 14 ml of Buffer A and disrupted
Ad@@O@'@TP
SO,,
‘@‘@
2M@
& 40'-
V
Frachons
1S5inVfroct,on)
Chart 1. dGTP-Sepharase column chromatography at ribonucleotide re
ductase derived tram MoIt-4F cells. Protein (17.3 mg) tram the ammanium
sulfate fractionation step (35 to 50%) was loaded an a dGTP-Sepharase
column (1.5 x 10.0 cm), and the column was eluted with Buffer B containing
various additives as shown. Fractions after dialysis against Buffer C were
analyzed far ribanucleatide reductase activities for CDP and ADP reductions
as described in the text. A, protein profile. B, the enzymeactivity of each
fraction assayed with an excess amount of Component B. Fractions 3 to 8
were pooled as Component A. C, the enzyme activity at each fraction
assayed with an excess amount at Component A. Fractions 27 to 31 were
pooled as Component B.
FEBRUARY1979
Downloaded from cancerres.aacrjournals.org on June 15, 2017. © 1979 American Association for Cancer Research.
437
C-H. Chang and V-c. Cheng
@
@
@
ponents A and B (data not shown). Each fraction from the
dGTP column chromatography was dialyzed overnight
against Buffer C and was assayed for both ADP and COP
meductase activity in the presence of excess amount of
either Component A on Component B. The results are
shown in Chart 1, B and C. Component A was eluted from
the column in the unabsorbed fractions, while Component
B could only be eluted with Buffer B containing 1 M KCI.
Fractions having the respective activity of each component
were combined. Component A was further purified as
described below. Component B was dialyzed against 2
changes of 40 volumes of Buffer C containing 30% sucrose
over a 6-hr period. The dialysate was further purified as
described below.
(A)
Addd,m (IC)
60
025M
JO.6
.@o4
4
Chromatographyof ComponentA on Blue Sepharose
Column.The pooledComponentA (10 ml) fromthe dGTP
Sepharose column was loaded on a blue Sephamosecolumn
(1.5 x 7.5 cm) previously equilibrated with Buffer C. The
column was then washed with the same buffer until the
absorbance of the eluant at 280 nm was less than 0.05, at
which point Buffer C containing 2 M KCI was applied. Each
fraction was dialyzed against Buffer C and then analyzed
for protein concentration and enzyme activity. More than
96% of the activity of Component A was not retained on the
column, as shown in Chart 2. The reason for retention of
the small amount of activity on the column (less than 4%) is
unknown at this time. However, it may be due to nonspecific
adsorption.
Chromatography of Component A on DEAE-Cellulose.
The combined fractions (17 ml) obtained from blue Sepha
nose chromatography were applied to a OEAE-cellulose
column (1.5 x 9 cm) previously equilibrated with Buffer C.
After loading Component A, the column was washed with 5
ml of the same buffer and was then eluted with Buffer C
containing 0.08 M, 0.25 M, and 2 M KCI, respectively. After
dialysis against Buffer C, fractions were analyzed for pro
tein concentration and Component A activity. The results
are shown in Chart 3A. Component A was retained on the
column and could be eluted with Buffer C containing 0.25
M KCI. The fractions
containing
Component
A activity
were
pooled for further purification.
Add'h@s
2.0
20
@
I
6
.6
12
1.2
8
0.8
4
o_4
4
Froction
8
2
(
85
6
.2
20
ml I fraction)
Chart 2. Chromatography at Component A on a blue sepharase column.
Component A (8.2 mg) from the dGTP-Sepharose column chromatography
was loadedon blue Sepharosecolumn (1.5 x 7.5 cm), and the columnwas
eluted with the solutions as indicated. After dialysis against Buffer C,
fractions were assayed for CDP and ADP reductase activities as described in
the text. Fractions 2 to 10 were pooled for further purification.
438
Fraction (I8mVfroction)
Chart 3. Chromatograph of Components A and B on DEAE-cellulose
columns. A , Component A (3.1 mg) from blue Sepharose column chromatog
raphy was loaded on a DEAE-cellulose column (1.5 x 9.0 cm), and the
column was eluted with Buffer C containing various additives as indicated.
Fractions after dialysis against Buffer C were assayed for CDP and ADP
reductase activities as described in ‘
‘Materials
and Methods.―Fractions 20
to 23 were pooled for further purification. In B, Component B (3.6 mg) from
dGTP-Sepharose column chromatography was loaded on a DEAE-cellulose
column (1.5 x 9.0 cm), and the column was eluted with solutions as
indicated. After dialysis against Buffer C, fractions were assayed for CDP
and ADP reductase activities as described in ‘
‘Materialsand Methods,―
Fractions 3 to 5 were pooled for further purification.
Chromatography of Component A on a Phenyl-Sepha
rose Column. Component A (7 ml), obtained from DEAE
cellulose chromatography, was made 1 M with respect to
ammonium sulfate and loaded onto a phenyl-Sepharose
column (1.5 x 12.5 cm) previously equilibrated with Buffer
C containing 1 M ammonium sulfate. The column was
washed with 5 ml of the same buffer and then eluted with
25 ml of Buffer C containing 25% ethylene glycol, followed
by 25 ml of Buffer C containing 50% ethylene glycol.
Fractions were collected and dialyzed against 40 volumes
of Buffer C for 3 to 4 hr. Each fraction was concentrated by
further dialysis overnight against 40 volumes of Buffer C
containing 45% sucrose. The volume of the concentrated
fraction was adjusted to 1 ml and analyzed for protein
concentration and enzyme activity as shown in Chart 4. The
activity of Component A was present in the fractions eluted
with 50% ethylene glycol.
Chromatography of Component B on DEAE-cellulose
Column. The pooled Component B (8 ml), after dGTP
Sephamose chromatography and dialysis against Buffer C
containing 30% sucrose, was loaded on a DEAE-cellulose
column (1.5 x 9 cm) previously equilibrated with Buffer C
containing 10% sucrose. The column was then washed with
12 ml of the same buffer followed by 15 ml of Buffer C
containing 2 M KCI. After dialysis against Buffer C, fractions
were analyzed for protein concentration and enzyme activ
ity. Component B activity was found in the void volume of
the column (Chart 3B).
Sedimentation of Component B by Sucrose Density
Gradient Centrifugation. The pooled Component B (4 ml)
obtained from the previous step was dialzyed overnight and
concentrated against 40 volumes of Buffer C containing
CANCERRESEARCHVOL. 39
Downloaded from cancerres.aacrjournals.org on June 15, 2017. © 1979 American Association for Cancer Research.
Two Ribonucleotide Reductase Components from Lymphoblast Cells
EthyleneGlycol 25
@
50
)%(
@
30
E
£
@
20
I
:
@
0.4
4
2
6
3
20
Frocton (tOrn)I fraction)
Chart 4. Chromatography of Component A an a phenyl-Sepharose cal
umn. Component A (1.4 mg) from DEAE-cellulose column chromatography
after addition of 1 M ammanium sulfate was loaded an a phenyl-Sepharose
column (1.5 x 12.5 cm) previously equilibrated with Buffer C containing 1 M
ammonium sulfate, and the column was eluted with 25 and 50% of ethylene
glycol in Buffer C as indicated. The volume at each traction after overnight
dialysis against Buffer C containing 45% sucrose was adjusted to 1 ml, and
each fraction was assayed tar ADP and CDP reductase activities as described
in “Materials
and Methods.―Fractions 13 to 15 were pooled.
@
30% sucrose. After removal of sucrose by Sephadex G-25
column chromatography, the dialysate (1 ml) was layered
onto an 11-mi linear sucrose gradient 5 to 20% (w/v)
prepared in Buffer C, and centrifuged at 100,000 x g for 20
hr at 2°in a SW 41 rotor. Fractions (0.9 ml) were collected
by puncturing the bottom of the tube and assayed for
Component B activity. The results are shown in Chart SC
and will be discussed under ‘
‘MolecularWeight Oetermina
tion.―
The pooled Component B obtained from dGTP-Sepha
rose chromatography following dialysis with 2 changes of
40 volumes of Buffer C was loaded on a blue Sephamose
column. The activity was retained on the column and could
be eluted with 1 M KCI (data not shown). However, the
specific activity of Component B after this chromatography
was not increased, due to the poor recovery of the activity
of Component B. Therefore, blue Sepharose chnomatogna
phy was omitted for purification of Component B.
In contrast to the behavior of Component A on a phenyl
Sepharose column, Component B came through the col
umn with unabsorbed proteins and with a recovery of less
than 10%. Therefore, this step also was not used for the
purification of Component B.
MolecularWeightDetermination
The molecular weight of nondissociated nibonucleotide
reductase (after the ammonium sulfate fractionation step),
as well as that of the purified A and B components, was
estimated by the method of sucrose density gradient. Sam
pIes (1 ml) were layered onto 11-ml linear sucrose gradients,
5 to 20% (w/v), prepared in Buffer C, and centrifuged at
100@000x g for 20 hr at 2°in a SW 41 rotor. Fractions (0.9
ml) were collected by puncturing the bottom of the tube
and were assayed for the enzyme activity. The results are
PK
‘(A)
80 @.
E
LDH
Hb
60
40
C
Commentson the Purificationof ComponentsA and B
@
@
@
The scheme used to purify both Components A and B of
mibonucleotide neductase derived from Molt-4F cells is sum
mamized in Table 1. The specific activity (pmol/min/mg
protein) of the final preparation of Component A was 384
for CDP reduction and 243 for ADP reduction. However, the
specific activity (pmol/mmn/mg protein) for the final prepa
ration of Component B was 669 for COP and 372 for ADP
reduction. The ratio of COP to ADP meductase activity is
1.73 ±0.19 throughout the purification procedure. Com
ponent A and Component B are both required to give the
enzyme activity; neither of them, by itself, has any detecta
ble catalytic activity. The addition of either component A or
Component B to the enzyme preparation obtained from
crude extract, streptomycin sulfate fractionation, or am
monium sulfate fractionation did not alter the enzyme
activity. The final preparations of Component A and Corn
ponent B are not homogeneous as judged by electropho
metic techniques. However, they were purified to such an
extent that nucleotide phosphatases and nucleoside di
phosphate kinases which would interfere with kinetic stud
ies of the enzyme were not present in the purified Compo
nents A and B (data not shown).
When Component B was applied to a OEAE-ceilulose
column, its activity was not retained on the column. Under
the same condition, Component A was absorbed to the
column (Chart 3). This observation suggested that Compo
nent B is relatively cationic as compared to Component A.
E
20
.
/@,
......(‘
I(B)
24- ‘@ 8
0
E
I
12
16
8
12
16
8
12
16
20
@20
C
0
U
16
12
a,
I
•.........S.. I4
a.
,@
0
20
I
20
24
20
24
‘(C)
@.
.
•..S....SSS..1@4
Bottom
I
Fraction (O.45 mI/fraction)
Chart 5. Ribanucleatide reductase activity profile after sucrose density
gradient centrifugatian. The sucrose density gradient centrifugatian condi
tions were performed as described in the text. Hemoglobin (Hb), lactate
dehydragenase (LDH), and pyruvate kinase (Pk) were used as markers. The
volume at each sample layered on the gradient was 1 ml. Fractions were
collected and assayed tar ADP and CDP reductase activities. A , the ribanu
cleatide reductase preparation obtained from the ammonium sulfate frac
tionatian (35 to 50%); B, Component A obtained from blue Sepharose
column chromatography; C, Component B obtained tram DEAE-cellulose
column chromatography.
FEBRUARY1979
Downloaded from cancerres.aacrjournals.org on June 15, 2017. © 1979 American Association for Cancer Research.
439
C-H. Chang and V-c. Cheng
Table 1
Purification of ribonucleotide reductasederived from human Mo!t-4Fcells
This representsthe purification of enzymesfrom 9 x 10@
cells.
(pmol/
activity0
mm)
StepProtein
CDPC
ADPCDP/ADPbUndissociated
(mg)Activity
enzyme@'Crudeextract114348
2.31.35Streptomycin
sulfate frac 57.72657
291.@9tionationAmmonium
sulfate
691.71tionationComponent
frac
17.32054
(pmol/min/mg)
ADPdSpecific
CDP
2663.1
169446
1197118
A1dGTP-Sepharose
chroma
91.67tographyBlue
8.2126
7615
Sepharose chroma
3.1183
351.69tographyDEAE-Cellulose
10959
chroma
1.4128
521.77tographyPhenyl-Sepharose
7392
chro
2431.@8matographyComponent
0.1247
30384
B°dGTP-Sepharose
chroma
3.6266
362.03tographyDEAE-cellulase
chrama
492.08tographySucrose
13073
1 .84 -1
87
901
density gradient0.16103
3721.80centrifugation
a pmal of substratereduced
02
60669
per mm per mg of protein
in the component
being
studied. An excessamount of one componentwas usedto determinethe specific activity
of the other component. This is to ensure that the latter component produces the
maximumactivity.
b The
ratio
of COP to ADP specific
activity.
C COP
was
used
as the
substrate.
The
detailed
procedure
of the
assay
is described
in
was
used
as the
substrate.
The
detailed
procedure
of
the
assay
is described
in
to
the
the text.
d ADP
the text.
t, The
addition
of
either
Component
A
or
Component
B
enzyme
preparation
obtained from crude extract, streptomycin sulfate fractionation, and ammonium sulfate
fractionation did not alter the enzymeactivity.
I Assays
were
performed
with
an excess
of Component
B as described
in ‘
‘
Materials
with
an
of
A
in
and Methods.―
0 Assays
were
@rformed
excess
Component
as
described
‘
‘Materials
and Methods.―
depicted in Chart 5. The apparent molecular weight was
estimated to be 210,000 for the nondissociated nibonucleo
tide neductase and 100,000 for both Component A and
Component B. The activities for both ADP and COP reduc
tase cosedimented in all studies.
Requirements for the Enzyme Activity
The requirements for the reductions of COP and AOP are
shown in Table 2. Like the nibonucleotide neductases de
nived from other sources (2, 13, 17-20, 22), the enzyme
obtained from MoIt-4F cells has a requirement for a specific
activator. ATP was required as an activator for COP reduc
tion, as was dGTP for ADP reduction. Magnesium ions and
OTT were also essential for COP and ADP reduction. OTT
was used in this study to substitute for thioredoxin reduc
tase which is a natural reducing protein (21). The optimal
concentration of OTT and MgCI2 for COP and ADP neduc
tase activity was 3 and 6 mM, respectively, in the presence
of activator at a concentration of 5 mM (data not shown).
440
Effectsof DivalentCationsand Polyamines
The requirements for divalent cations and polyamines for
either AOP or COP meductase activity were examined by
using a mixture of purified A and B components. The
results are presented in Table 3. No reaction took place in
the absence of divalent cations. Among the divalent cations
and polyamines tested at a concentration of 6 mM, MgCI2
gave the highest velocity for both AOP and COP reduction.
MgSO4 gave the same velocity as MgCl2 for COP reduction
but not for AOP reduction. This result was due to the
substitution of the Cl anion by the SO@= ion. Ca2@could
replace Mg2@and maintain full activity for CDP reduction
but resulted in lower activity for AOP reduction. Mn2@could
partially substitute for Mg2@for COP reduction, but not for
AOP reduction. Zn2@and Fe2@could not substitute for Mg2@
for either AOP or COP reduction. Spenmidine and spermine
at the same concentration as MgCl2 gave 70% activity for
COP reduction and 30 to 40% activity for AOP reduction as
compared to the rate seen when MgCl2 was used. Further
CANCERRESEARCHVOL. 39
Downloaded from cancerres.aacrjournals.org on June 15, 2017. © 1979 American Association for Cancer Research.
Two Ribonucleotide ReductaseComponents from Lymphoblast Cells
Table 2
reductionThe
Requirementsfor COPand AOP
reductionwas
complete incubation mixture for COP and ADP
theseassays,
the sameas described in ‘
‘Materials
and Methods.'‘
For
thepurification
ComponentsA and B from the respectivelast steps of
(w/w),respectively.
procedurewere combined in the ratio of 1.4:1.0
The maximal activity with the reconstituted nibonucle
otide
and12reductasewas equal to 20 pmol of COPreduced per hr
pmol of ADPreduced per hr.%
activityCOP
Total
tionNone
Componentomitted
100Activator
4MgCI2
(ATP or dGTP)
reduction
100
ADPreduc
4
4DTT
5
1Enzyme
2
0
Table 4
Reduction
of COP and ADP by ribonucleotide
when
The amount
of Component
A used was fixed
at 6 pg/assay.
ComponentsA and B usedfor theseassayswere obtained from the
last respectivestepsof
procedure.CDP
the purification
neductase AOPreductase
Component
B
activity
(pmol/
activity (pmol/
addedCDP/ADP―0
(/.Lg)
hr)
0
6
4.6
hr)
0
2.7
0
1.70
11
8.5
5.1
1.67
16
21
9.5
10.8
5.7
6.4
1.67
1.69
of CDP to ADP reductase
activity.
a The ratio
0
reductase
ComponentA was titrated by ComponentB
DISCUSSION
When streptomycin sulfate was added to the crude ho
mogenate to remove nucleic acid contaminants, the total
enzyme activity for AOP and COP reduction increased about
7-fold (Table I ). In view of the report by Cory et a!. (11) that
reduction catalyzed by ribonucleotide
reductase derived from
Molt-4Fcells
RNA and oligomibonucleotides markedly inhibit nibonucleo
Purified reconstituted ribonucleotide reductasewas used which tide reductase activity, it seems plausible that this observa
gave an activity of 60 pmol/hr reduction of COP in the standard tion was due to the precipitation of these inhibitors by the
assayconditions. SeeTable 2 for a description of the reconstituted streptomycin sulfate. In addition, as suggested by Cohen et
ribonucleotide
reductase.
a!. (9), there may be some competitive endogenous precun
activity―COP
sons, formed from breakdown of DNA and ANA, which are
removed during the subsequent dialysis of the streptomycin
reduction@'None00MgCI,100100MgSO49563MnCl,350CaCI29058FeSO400ZnSO400Putrescine3320Spermidine6739Spemmine7130
reductionb ADP
Addition
(6 mM)%of
sulfate pellet.
The observation that the nibonucleotide reductase from
Molt-4F cells was a cytoplasmic enzyme is in agreement
with other published work on the mammalian enzyme (15,
19).
Efforts to purify nibonucleotide reductase from Molt-4F
cells have resulted in the separation of 2 components (A
and B). After the 2 components of nibonucleotide reductase
were dissociated by dGTP-Sepharose chromatography, a
a Percentage of activity was calculated by comparing the activity
substantial loss of activities of Components A and B was
under different conditions with that found with 6 mMMgCI2.
observed when these activities were assayed using an
b The
assay
for COP
reduction
was
the same
as described
in
excess amount of Components B and A, respectively (Table
“Materials
and Methods.―
1). This might be due to the fact that, when Components A
C The
assay
for
ADP
reduction
was
the
same
as
described
in
and B were reconstituted under the assay conditions used,
“Materials
and Methods.―
they did not assume their native conformation. This may be
more, in the presence of 6 mM MgCl,, Mn2@and Zn2@had supported by the observation that, when purified Compo
strong differential inhibitory effects on both COP and AOP nents A and B were mixed and centrifuged in sucrose
reduction (data not shown).
density gradient, no activity was observed with the same
sedimentation rate as that of nondissociated nibonucleotide
Cellular Localizationof the Enzyme
meductase(data not shown).
Molt-4F cells in the log phase of growth were fractionated
Some properties of the Components A and B were re
into subcellulam fractions according to the procedure de
vealed by their behavior during the process of purification.
scnibed previously (8). More than 95% of the activity for The 2 components have similar apparent molecular weights
both ADP and COP reduction was present in the cytosol
which are comparable to those for the 2 active components
from Ehrlich tumor cells (12). This is in contrast to 2
fraction, only 1 to 3% of the activity was associated with the
nuclear fraction, and no detectable activity was found in subunits form Escherichia co!i which have molecular
either the mitochondnial on the endoplasmic reticulum frac
weights of 160,000 (protein B1) and 78,000 (protein B2) (2,
tions.
26). Component A is relatively anionic and hydrophobic
when compared with Component B. The binding sites for
Titrationof ComponentA by ComponentB
tniphosphate nucleotides, which could serve as either acti
Table 4 shows the titration of Component A by Compo
vators or inhibitors, appear to be present on Component B,
nent B. The ratio of COP to AOP reductase activity was based on the observation that only Component B has
constant at any ratio of Component B to A tested.
binding affinity for dGTP-Sepharose and blue Sepharose.
Table 3
Effects of the substitution of MgCI2by variouschloride and sulfate
forms of divalent cations and polyamineson ADP and COP
FEBRUARY1979
Downloaded from cancerres.aacrjournals.org on June 15, 2017. © 1979 American Association for Cancer Research.
441
C-H. Chang and V-c. Cheng
Mg2@may enhance the association of Component A and
Component B, because no clear resolution of the 2 corn
ponents was obtained when the undissociated enzyme was
applied to a dGTP-Sephanose column in the presence of
[email protected] requirement for Mg2@in the binding of Subunits B1
to B2 of nibonucleotide neductase derived from E. coli has
been reported previously (4).
Mg2@is the most effective divalent cation among all those
tested to fulfill both ADP and COP meductase activity.
Replacement of MgCI2 in the reaction mixture by various
cations, or addition of various divalent cations to the
reaction mixture containing 6 mM MgCl,, has different
effects on the ADP and COP reduction catalyzed by the
reconstituted enzyme. The different effect of MgCl, and
MgSO4 on COP and ADP enzyme activity must relate to the
difference in the anions (Cl versus SO4) . Unlike the
enzymes for other mammalian systems, which require low
concentration of exogenous ferrous or femnicion for optimal
activity (18, 19, 22), addition of Fe2@will not alter COP and
AOP meductaseactivity in Molt-4F cells. In the presence of 6
mM MgCI,,
Mn2@ and
Zn2@ have
inhibitory
effects
that
are
more pronounced on AOP reduction than on COP reduction
(data not shown). Cohen and Banner (9) reported that, in
the absence of MgCI2, the enzyme reduction system from
T6n@-infected E. coli could be stabilized or activated by
polyamines. It has been observed in this laboratory that
polyamines could partially replace Mg2@in fulfilling the
metal ion requirements. On the contrary, in the presence of
MgCl, at 6 mM, none of the polyamines at concentrations
higher than 0.4 mM tested were demonstrated to stimulate
either COP or ADP meductaseactivity (data not shown).
The differences observed in the sensitivity of AOP and
COP reduction to various agents may be explained in 2
ways. AOP and COP neductase activities might reside in 2
separate enzyme entities, or they might exist in the same
enzyme but have different active sites. The following obser
vations tend to support the concept that the 2 neductase
activities are associated with the same molecule: (a) both
COP and ADP reductase activities remain associated
throughout the purification with a constant ratio; (b) the
rate of reduction of COP and AOP flucttiates similarly
throughout the HeLa cell cycle (7) [this observation is
different from the results reported by Peterson and Moore
using Chinese hamster fibnoblast cells (24)]; and (C) the
ratio of COP to ADP reductase activity was the same at any
tested ratio of Component B to A.
Assay of Proteins
2.
3.
4.
5,
6.
7.
8.
9.
Deaxyribasyl-synthesizing
1 1 . Cory,
13.
14.
15.
16.
17.
18.
19.
20.
21.
22.
25.
REFERENCES
27.
442
P.,
Stein,
S.,
Dairman,
W.,
and
Udenfriend,
5.
Fluarametric
System tram T6r'-intected
Escherichia
coli.
J. G. Inhibition
of Ribanucleotide
Reductase
from
Ehrlich
Tumor
Cory,
J.
G.,
Mansell,
M.
M.,
and
Whitfard,
T.
W.,
Jr.
Control
of
Ribonucleatide Reductase in Mammalian Cells. Advan. Enzyme Regula
tion, 14: pp. 45—62,
1975.
26.
1 . BOhIen,
Biophys.,
Cells by RNA. Cancer Res., 33: 993-998, 1973.
12. Gary, J. G., Fleischer, A. E., and Munro, J. B., III. Reconstitution of the
Ribonucleatide Reductase Enzyme tram Ehrlich Tumor Cells. J. Bial.
Chem., 253: 2898-2901 , 1978.
24.
We wish to thank Joanne Cobler, Linda Roberts, and Susan Grill far their
excellent technical assistance and to express our appreciation to Dr. Dennis
R. Conrad far his valuable advice and comments.
Range. Arch, Biochem.
J. Biol. Chem., 237: 1376—1378,
1962.
10. Collins, T., David, F., and Van Lancker, J. L. CDP and ADP Reductase in
Rat Regenerating Liver. Federation Proc., 31: 641, 1972.
23.
ACKNOWLEDGMENTS
in the Nanogram
155: 213—220,
1973.
Brown, N. C., Canellakis, Z. N., Lundin, B. , Reichard, P., and Thelander,
L. Ribanucleaside Diphosphate Reductase. Purification of the Two
Subunits, Protein B1 and B2. European J. Biachem., 9: 561-573, 1969.
Brawn, N. C. , Eliassan, R. , Reichard, P., and Thelander, L. Spectrum
and Iran Content at Protein B2 from Ribonucleaside Diphasphate
Reductase. European J. Biochem., 9: 512-518, 1969.
Brown, N. C., Larssan, A., and Reichard, P. On the Subunit Structure of
Ribanucleaside Diphosphate Reductase. J. BioI. Chem. , 242: 42724273, 1967.
Chang, C-H., and Cheng, V-C. Purification and Characterization of
Human Ribonucleotide Reductase. Pharmacologist, 19: 135, 1977.
Chang, C-H., and Cheng, V-C. Properties of Ribonucleatide Reductase
Isolated from a Human Lymphoblast Line (Malt 4F). Proc. Am. Assoc.
Cancer Res., 19: 68, 1978.
Cheng, V-C., Chang, C-H., Williams, M. V. , Cass, C. E. , and Paterson,
A. R. P. Fluctuations at Ribonucleatide Reductase Activity and Deoxyri
bonucleatide Pools in Synchronized HeLa Cells. Proc. Am. Assoc.
Cancer Res., 18: 185, 1977.
Cheng, Y-C. , and Ostrander, M. Deaxythymidine Kinase Induced in HeLa
TK Cells by Herpes Simplex Virus Type I and Type II. J. Biol. Chem.,
251: 2605-2610, 1975.
Cohen, S. S., and Barner, H. D. Spermidine in the Extraction of the
Cory,
J. G.,
Russell,
F. A., and Mansell,
M. M. A Convenient
Assay
for
ADP Reductase Activity Using Dawex-1-Borate Columns. Anal. Bio
chem., 55: 449-456, 1973.
Elford, H. L. Subcellular Localization of Ribanucleatide Reductase in
Navikaff Hepatoma and Regenerating Rat Liver. Arch. Biochem. Bia
phys., 155: 213-220, 1973.
Hoffmann, P. J., and Blakley, R. L. An Affinity Adsorbent Containing
Large Scale Preparation of Ribanucleatide Reductase of Lactobacillus
Ieichmannii. Biochemistry, 14: 4804—4812,
1975.
Halmgren, A., Reichard, P., and Thelander, L. Enzymatic Synthesis of
Deaxyribanucleatides, VIII. The Effects of ATP and dATP in the CDP
Reductase System from E. coli. Proc. NatI. Acad. Sci. U. S., 54: 830836, 1965.
Hopper, S. Ribanucleotide Reductase at Rabbit Bane Marrow. 1. Purifi
cation, Properties, and Separation into Two Protein Fractions. J. Biol.
Chem., 247: 3336-3340, 1972.
Larssan, A. Ribanucleotide Reductase from Regenerating Rat Liver.
EurapeanJ.Biochem.,11:
113-121,1969.
Larsson, A., and Reichard, P. Enzymatic Synthesis of Deaxyribanuclea
tides. IX. Allosteric Effects in the Reduction of Pyrimidine Ribonuclea
tides by the Ribanucleaside Diphosphate Reductase System of Esche
richia coIl. J. Biol. Chem., 241: 2533-2539, 1966.
Laurent, T. C., Moore, E. C., and Reichard, P. Enzymatic Synthesis at
Deaxyribanucleatides. IV. Isolation and Characterization of Thioredoxin,
the Hydrogen Donor from Escherichia coli, B. J. Bial. Chem., 239: 34363444, 1964.
Moore, E. C. [21] Mammalian Ribanucleaside Diphosphate Reductase
Methods Enzymal., 12: 155-164, 1967.
Moore, E. C. Components and Control at Ribonucleatide Reductase
System of the Rat, Advan. Enzyme Regulation, 15: 101-114, 1976.
Peterson, M. D., and Moore, E. C., Independent Fluctuations of Cytidine
and Adenasine Diphasphate Reductase Activities in Cultured Chinese
Hamster Fibroblasts. Biachim. Biophys. Acta, 432: 80-91 , 1976.
Steeper, J. R., and 5teuart, C. D. A Rapid Assay for CDP Reductase
Activity in Mammalian Cell Extracts. Anal. Biachem., 34: 123-130, 1970.
Thelander, L. Physicachemical Characterization of Ribanucleaside Di
phosphate Reductase from Escherichia coIl. J. Bial. Chem., 248: 45914601, 1973.
Thelander, L. Reaction Mechanism of Ribanucleaside Diphasphate
Reductase tram Escherichia coli. J. Bial. Chem., 249: 4858-4862, 1974.
CANCERRESEARCHVOL. 39
Downloaded from cancerres.aacrjournals.org on June 15, 2017. © 1979 American Association for Cancer Research.
Demonstration of Two Components and Association of
Adenosine Diphosphate-Cytidine Diphosphate Reductase from
Cultured Human Lymphoblast Cells (Molt-4F)
Chi-Hsiung Chang and Yung-chi Cheng
Cancer Res 1979;39:436-442.
Updated version
E-mail alerts
Reprints and
Subscriptions
Permissions
Access the most recent version of this article at:
http://cancerres.aacrjournals.org/content/39/2_Part_1/436
Sign up to receive free email-alerts related to this article or journal.
To order reprints of this article or to subscribe to the journal, contact the AACR Publications
Department at [email protected].
To request permission to re-use all or part of this article, contact the AACR Publications
Department at [email protected].
Downloaded from cancerres.aacrjournals.org on June 15, 2017. © 1979 American Association for Cancer Research.