[CANCER RESEARCH 42, 4321-4324,
0008-5472/82/0042-OOOOS02.00
November 1982]
Characterization of an Adenosine S'-Triphosphate- and Deoxyadenosine 5'Triphosphate-activated Nucleotidase from Human Malignant Lymphocytes1
Dennis A. Carson and D. Bruce Wasson
Department of Clinical Research, Scripps Clinic and Research Foundation, La Jolla, California 92037
ABSTRACT
The kinetic properties of a soluble, magnesium-dependent
5'-nucleotidase from human malignant lymphocytes have been
determined. The partially purified enzyme is distinct from
plasma membrane-associated
5'-nucleotidase and is free of
nonspecific phosphatase activity. Among purine ribonucleotides, it reacted efficiently with inosine 5'-monophosphate and
guanosine 5'-monophosphate
and to a lesser degree with
deoxyguanosine 5'-monophosphate. Adenosine 5'-monophosphate and deoxyadenosine 5'-monophosphate
were 30-fold
less efficient substrates. Increasing concentrations of adenosine S'-triphosphate and deoxyadenosine 5'-triphosphate from
0 to 3 rriM enhanced 5'-nucleotidase
activity up to 7-fold.
Guanosine 5'-triphosphate
and deoxyguanosine
5'-triphosphate were much less effective enzyme activators, while uridine
S'-triphosphate was without effect. Inorganic phosphate in
hibited dephosphorylating activity in both adenosine 5'-triphosphate-supplemented and unsupplemented buffer. The activa
tion of this 5'-nucleotidase by deoxyadenosine 5'-triphosphate,
combined with the relative inability of the enzyme to dephosphorylate deoxyadenosine
5'-monophosphate,
conceivably
may contribute to the adenine nucleotide degradation induced
by deoxyadenosine in normal and malignant lymphocytes.
INTRODUCTION
Intracellular nucleotide degradation in human cells is highly
regulated (10). However, the exact enzymes catalyzing the
dephosphorylation
of purine 5'-monophosphates
have not
been well characterized. The most abundant human 5'-nucleotidase [5'-ribonucleotide
phosphohydrolase (EC 3.1.3.5)] is
on the external surface of the plasma membrane and probably
plays no role in intracellular nucleotide degradation (2, 5, 18,
19).
Recently, several investigators have described a novel 5'nucleotidase in the cytoplasm of rat liver and chicken liver that
is distinct from the plasma membrane enzyme (11 -13, 24-26).
The activity of the cytosolic enzyme was enhanced by ATP at
concentrations that inhibited plasma membrane 5'-nucleotidase. Several lines of evidence suggested that in rat liver the
ATP-stimulated cytoplasmic nucleotidase was a predominant
enzyme catalyzing the dephosphorylation of purine nucleotides
(24-26).
In the presence of an inhibitor of adenosine deaminase,
nondividing human lymphocytes exposed to /¿M
concentrations
of deoxyadenosine in vitro or in vivo progressively accumulate
' Supported by Grants GM 23200 and CA 31497 from the USPHS.
Received April 26. 1982; accepted July 21, 1982.
NOVEMBER
1982
dATP (5, 14, 19, 23). Subsequently, ATP levels, and indeed
the total intracellular pools of adenine nucleotides, slowly de
cline (1,5,19).
The individual enzymes potentially catalyzing
nucleotide degradation in lymphocytes with elevated dATP
levels have not been characterized. Recently, an ATP- and
dATP-stimulated
IMP-dephosphorylating
activity was de
scribed in crude extracts of a human T-lymphoblastoid cell line
(1).
In the present investigations, we have partially purified from
malignant human lymphocytes a soluble nucleotidase with
properties analogous to the rat and chicken liver enzymes. The
kinetics of the enzyme has been determined, with special
reference to the effects of adenine deoxynucleotides as sub
strates and activators.
MATERIALS
AND METHODS
Enzyme Purification.
Splenic tissue, largely replaced by a well-
differentiated lymphocytic lymphoma, was removed during surgery and
then frozen at -70° for 3 weeks. 5'-Nucleotidase
was purified from
the rapidly thawed specimen, following the method developed by Itoh
(11), except that the second phosphocellulose column was eluted with
a linear gradient of 200 to 800 mw NaCI instead of with ATP and the
low-ionic strength precipitation step was omitted. When stored as
described by Itoh (11), the enzyme was stable at -70° for at least 1
month.
5'-Nucleotidase Assay. 5'-Nucleotidase activity was determined
radiochemically using [8-14C]IMP as substrate and by measuring the
release of inorganic phosphate from various nucleoside 5'-monophosphates, exactly as described earlier (4). Unless stated otherwise, the
buffer was 100 mw imidazole-HCI (pH 6.5), 50 rnw MgCI2, 500 mw
NaCI, 0.1% bovine serum albumin, containing varying concentrations
of nucleoside 5'-monophosphate,
and effectors as indicated. The re
actions were initiated by the addition of 1 to 5 HQenzyme protein and,
after 15 to 45 min at 37°, were terminated by heating to 100° for 1
min. For the radiochemical assay, inosine was separated from nucleo
tides by chromatography on polyethyleneimine-cellulose
jn methanol:
water (1:1) in the presence of appropriate standards (4). The nucleosides were visualized under UV, cut out, and counted in a liquid
scintillation spectrometer. Enzyme activities are expressed as nmol
product per min per mg protein and were linear with protein concentra
tion and with time for the data reported. Protein content was determined
by the method of Lowry ef al. (18), using bovine serum albumin as a
standard.
Materials. All common nucleosides and nucleotides were purchased
from Sigma Chemical Co. (St. Louis, Mo.). The [8-14C]IMP (50 mCi/
mmol) came from Amersham/Searle
Corp. (Arlington Heights, III.) and
was purified by high-performance
liquid chromatography.
ATP and
GTP were purified immediately before use by DEAE-cellulose chro
matography, using a 100 to 500 ITIMNaCI gradient in 5 mw potassium
phosphate, pH 5.0, and contained less than 2 nmol orthophosphate
per /imol nucleotide. Other materials were of the highest grade com
mercially available.
4321
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D. A. Carson and D. B. Wasson
RESULTS
Enzyme Purification. Because the IMP-dephosphorylating
activity in the crude lymphocyte extract (5.0 nmol/min/mg
protein) represented a mixture of nucleotidases and phosphatases, it was not possible to determine the exact purification
factor or yield of the 5'-nucleotidase. However, the estimated
cytosolic nucleotidase activity in lymphocytes ranged from 0.4
to 1.0 nmol/min/mg protein. The final enzyme preparation was
free of detectable ß-glycerophosphate- and ATP-dephosphorylating activities (<0.5 nmol dephosphorylated/min/mg
pro
tein at a substrate concentration of 40 ITIM)(4). It also lacked
measurable adenosine deaminase or purine nucleoside phosphorylase (3). At an IMP concentration of 3 mM in the standard
buffer lacking ATP, the specific activity of the partially purified
enzyme was 1.4 /imol/min/mg
protein. The total yield from
14.2 starting material was 2.7 mg protein, containing 3.78
jumol IMP-dephosphorylating
activity per min.
Effect of pH and MgCI2. When assayed in imidazole-HCI
buffer with IMP as substrate, the pH optimum of the enzyme
was 6.4 (Chart 1). At neutral pH, 5'-nucleotidase activity was
approximately 50% maximal.
The enzyme activity was not inhibited by 10 mM tartrate or
5 mM jS-glycerophosphate. Ten mM fluoride inhibited enzyme
activity by 65%. In the absence of added MgCI2, no IMPdephosphorylating activity was measurable.
Substrate Specificity. The lymphocyte 5'-nucleotidase dephosphorylated all 8 purine and pyrimidine 5'-monophosphates
studied (Table 1). Over the range of concentrations
dGMP
AMP
4
5
Substrate(mM)
Chart 2. Relation of substrate concentration to enzyme velocity. The reaction
conditions were the same as in Chart 1 except that varying concentrations of
IMP, dIMP, QMP, dGMP, and AMP were used.
Il
tested,
0.4
10
20
30
Substrate (mM)
40
50
Chart 3. Relation of substrate concentration to enzyme velocity and effect of
ATP. The reaction conditions were as described in Chart 1 except that varying
concentrations of AMP and dAMP were used, insert, effect of 3 mM ATP upon
the rate of the reactions.
0.1
5.5
6
6.5
7.5
pH
Chart 1. Effect of pH on 5'-nucleotidase activity. Enzyme activity was assayed
in 100 mM imidazole-HCI containing 50 IÕIMMgCI.. 0.1% bovine serum albumin,
2 mM IMP, and 1.5 «gprotein in a total volume of 100 »IProduct formation was
determined after 45 min at 37" by the inorganic phosphate method.
Table 1
Substrate specificity of the ¿'-nucleotidase
for each nucleotide, product formation was determined by the release of
inorganic phosphate at substrate concentrations from 0.1 to 100 mM. The
reaction buffer lacked ATP or inorganic phosphate. The V,,,,. values for the
respective nucleotides are shown relative to IMP, for which the specific activity
was 1.4 ;imol/min/ mg protein. The S •¿
is the substrate concentration at which
the velocity was 0.5 V,.,,. and is reported for the inefficient substrates AMP,
dAMP, and dCMP, which did not follow Michaelis-Menten kinetics. The ratios of
V,„„/K„„
or V,,,,,/S.,... shown in parentheses, yielded estimates of the overall
efficiency of the enzyme toward the individual substrates and are compared to
IMP.
substrate-velocity
plots were hyperbolic with the preferred
substrates IMP, dIMP, GMP, dGMP (Chart 2), and CMP (not
shown). Linear regression analysis of Eadie-Hofstee plots of
the data by the method of least squares yielded r* values
>0.90 in each case.
With the inefficient substrates AMP and dAMP (Chart 3),
substrate-velocity plots were approximately linear at concen
trations up to 12 mM. Maximal enzyme activity was approached
only at nucleotide concentrations of 30 to 50 mM, far above
any achievable physiological range. With the latter 2 sub
strates, the nucleotide concentration at which dephosphorylating activity was half-maximal (S0 5) O 5) was used to estimate
overall substrate efficiency by the ratio Vmax/S05. For the other
substrates, the ratio Vmax/Kmwas used (16). As shown in Table
1, the enzyme reacted most efficiently with IMP, dIMP, and
GMP; dGMP was dephosphorylated
5-fold less efficiently.
When compared to IMP and dIMP, AMP and dAMP were 30-
or Sos
SubstrateIMPdIMPAMPdAMPGMPdGMPCMPdCMPRelative
Vâ„¢,1.001.060.910.901.050.720.520.57Km
(mM)0.570.62>15>150.642.155.98>15Relative
efficiency10.98<0.03<0.030.940.190.05<0.002(1.75)(1.71)«0.06)«0.06)(1
fold less efficient substrates.
Effect of ATP, dATP, GTP, dGTP, and Inorganic Phos
phate. With 200 juM IMP as substrate, increasing concentra
tions of ATP or dATP from 0 to 3 mM increased dephosphory.64)(0.33)(0.087)«0.04)
lating activity up to 7-fold (Chart 4). GTP and dGTP were poor
but detectable activators of the enzyme, while UTP barely
augmented IMP dephosphorylation.
ATP and dATP also en-
4322
CANCER
RESEARCH
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VOL. 42
dATP-activated
Nucleotidase
in Lymphocytes
0.6
developed by Itoh (11) for the rat liver cytosolic nucleotidase
effectively separated the human enzyme from other dephos-
0.5
phorylating activities.
Among purine nucleotides, the preferred substrates for the
enzyme were IMP, dIMP, GMP, and dGMP. When compared to
IMP, both AMP and dAMP were 30-fold less efficient sub
strates. At optimal pH and magnesium concentrations, the net
dephosphorylating activity of the enzyme was modulated pri
marily by the relative concentrations of substrate, ATP, and
inorganic phosphate.
Previous kinetic analyses of mammalian cytosolic nucleoti
dases did not examine in detail the effects of dATP and dGTP
on IMP dephosphorylation,
although dATP was reported to
stimulate the chicken liver enzyme (12). We found that dATP
was as effective as ATP in enhancing IMP dephosphorylation
by the 5'-nucleotidase from human malignant lymphocytes.
Indeed, even 100 /ÕMdATP significantly augmented IMP-de
phosphorylating activity in a buffer containing 3 rriM ATP and
2 mM phosphate. By comparison, GTP and dGTP were far less
potent activators of the enzyme.
As noted earlier, although dATP activated the nucleotidase,
dAMP was an inefficient substrate for the enzyme. In these 2
aspects, the kinetics of the nucleotidase strongly resemble
human erythrocyte adenylate deaminase (1, 17). The latter
enzyme also reacts inefficiently with dAMP but is stimulated
markedly by both ATP and dATP. The 2 enzymes, adenylate
deaminase and 5'-nucleotidase, are probably pivotal in regu
0.4
0.3
GTP
dorp
0.2
0.1
0123
NucleosidetriphosphateImMl
Chart 4. Activation of 5'-nucleotidase by ATP, dATP, GTP. dGTP, and UTP.
The reaction mixtures contained 200 /IM [8-'4C]IMP (0.03 jiCi) and 0.5 fig protein
in 100 /il of the standard buffer. After 20 min. inosine was separated from IMP by
thin-layer chromatography.
Point, mean of 2 experiments, each performed in
duplicate: bars, S.E.
0.1
0.6.
I
0.4
ÃŽ0.2
01234
Phosphate
ImMl
Chart 5. Inhibition of 5'-nucleotidase by inorganic phosphate. The reactions
contained 200 fiM [8-'4C]IMP (0.03 /iCi) and 1.5 jig protein in a standard reaction
buffer containing 3 ITIM ATP (•)or lacking ATP (O). Product formation
assayed radiochemically.
was
hanced the rate of dephosphorylation of AMP and dAMP (Chart
3, inset). However, even in a buffer containing 3 ITIMATP, the
rate of dephosphorylation of AMP and dAMP was an order of
magnitude less than the rate for IMP or GMP.
Inorganic phosphate at concentrations from 0 to 4 mM sub
stantially reduced IMP-dephosphorylating
activity. The inhibi
tion was significant in both ATP-supplemented and unsupplemented buffers (Chart 5).
Chart 6 shows the effects of increasing concentrations of
dATP, ATP, and dGTP upon enzyme activity in a buffer con
taining 0.2 rriM IMP, 3 HIM ATP, 0.5 rriM GTP, and 2 ITIM
inorganic phosphate. Notably, as little as 100 JUMdATP in
creased IMP dephosphorylation significantly. Those concentra
tions of dATP are achieved routinely in normal or malignant
lymphocytes incubated with deoxyadenosine and the adenosine deaminase inhibitor deoxycoformycin,
vivo (4, 19).
lating the overall degradation of adenine ribonucleotides in
human cells (1, 6, 10, 22, 24-26). The activation of both
enzymes by dATP, combined with their relative inability to
metabolize dAMP, could contribute to adenine nucleotide deg
radation in adenosine deaminase-inhibited human lymphocytes
exposed to deoxyadenosine (1,5,19).
Under such conditions,
¡ntracellular dATP levels may reach nearly 30% of the total
ATP content (3).
In preliminary experiments, we have also purified and char
acterized an ATP-stimulated nucleotidase from extracts of nor
mal resting human peripheral blood lymphocytes. However, no
detectable ATP-regulated nucleotidase has been recovered
from human erythrocytes. The lack of an ATP- or dATP-acti
vated nucleotidase in human erythrocytes may explain why
both in vitro and in
ÃŽ .28
DISCUSSION
This study presents evidence demonstrating that human
malignant lymphocytes contain a soluble, magnesium-depend
ent nucleotidase whose activity is enhanced by ATP and is
inhibited by inorganic phosphate. Our earlier attempts to char
acterize cytosolic nucleotidases in crude extracts of human
tissues were hampered by interference from the more abundant
plasma membrane-associated 5'-nucleotidase, as well as from
nonspecific phosphatases. Fortunately, the purification method
NOVEMBER
1982
0 100 200 400 600 800 1000
Nucleoside
Triphosphate
luMi
Chart 6. Effect of dATP, ATP. and dGTP upon 5'-nucleotidase
activity in a
buffer containing 3 mM ATP, 0.5 mM GTP, 2 mM phosphate, and 0.2 mM IMP.
Point, mean for 2 experiments, each performed in duplicate; bars, S.E.
4323
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D. A. Carson and D. B. Wasson
dATP rises to higher levels in erythrocytes than lymphocytes,
before ATP degradation ultimately ensues (4, 19, 23).
Since neither AMP deaminase nor the ATP-stimulated 5'nucleotidase effectively degrade dAMP, other dAMP-dephosphorylating enzyme(s) must exist in the cytoplasm of human
cells. In support of this notion, the cumulative evidence of
several laboratories indicates that mammalian cells actually
contain at least 3 separate nucleotidases: (a) the soluble nucleotidase described herein that reacts preferentially with IMP
and GMP and is stimulated by ATP (11-13, 24-26); (b) a
soluble nucleotidase that reacts preferentially with deoxynucleotides, including dAMP, independent of ATP levels (4, 8, 9);
and (c) a plasma membrane-associated nucleotidase that effi
ciently dephosphorylates AMP and dAMP and which is in
hibited by ATP (2, 4). Most workers now agree that the plasma
membrane enzyme plays no significant role in intracellular
purine nucleotide degradation (4, 7, 20, 21). The markedly
different substrate specificities of the 2 described intracellular
nucleotidases is consistent with the view that lymphocytes
regulate purine ribonucleotide and deoxyribonucleotide catabolism via different mechanisms. Indeed, considering the diver
gent functions of the 2 classes of purine nucleotides for cell
growth and metabolism, this conclusion is not unexpected.
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CANCER
RESEARCH
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VOL.
42
Characterization of an Adenosine 5′-Triphosphate- and
Deoxyadenosine 5 ′-Triphosphate-activated Nucleotidase from
Human Malignant Lymphocytes
Dennis A. Carson and D. Bruce Wasson
Cancer Res 1982;42:4321-4324.
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