[CANCER
RESEARCH
26, 2213-2217, October
The Antitumor
1966)
Activity of Escherichia
coli L-Asparaginase1
JOSEPH ROBERTS, MORTON D. PRAGER, AND NICHOLAS BACHYNSKY
Wadley Research Institute and Ulnari Bank and The, Graduate Research Institute of Baylor University, Dallas, Texas
Summary
Two L-asparaginase components were isolated from Escherichia
coli by diethylaminoethyl cellulose column chromatography.
The early emerging comjxment (Peak I L-asparaginase), capable
of causing complete regression of the Gardner 6C3HED lymphosarcoma, exhibited optimum activity at pH 7.5-8.6 and was
stable on prolonged incubation with C3H mouse serum or perito
neal ascitic fluid. In contrast, the Peak II L-asparaginase com
ponent exhibited optimum activity at about pH 8.5, was sig
nificantly inactivated on incubation with the mouse humoral
fluids, and lacked tumor-inhibitory activity. A number of factors
which may influence the therapeutic usefulness of enzymes are
presented.
Introduction
Guinea pig serum (GPS) has been shown to inhibit a number
of transplantable lymphomas in mice and rats as well as certain
spontaneous and radiation-induced leukemias in mice (1, 6, 7).
Broome (2, 3) has presented evidence that the antitumor prin
ciple of GPS is L-asparaginase. This suggestion has been con
firmed by Yellin and Wriston (11), who demonstrated that a
purified preparation of GPS a.sparaginase which was homogene
ous by ultracentrifugation and electrophoresis had antitumor
activity comparable to that of guinea pig serum. However, with
asparaginase preparations from various other sources, certain
anomalies with regard to antitumor activity have been un
covered. For instance, Suld and Herbut (10) showed that L-as
paraginase obtained from guinea pig liver possessed only a
fraction of the antitumor activity of the GPS enzyme. Further
more, Mashburn and Wriston (8) reported that although a
highly purified E. coli L-asparaginase preparation possessed
good antitumor activity, a similar enzyme preparation obtained
from Bacillus coagulan»did not inhibit tumor growth. This
report describes the isolation from E. coli of 2 forms of L-as
paraginase, only 1 of which possesses antitumor activity. Some
properties of these 2 preparations which might account for the
difference in antitumor activity will be presented.
Materials
and Methods
PREPARATION
OFENZYME.Escherichia coli strain 11303 (Ameri
can Type Culture Collection) was grown at 37°Cwith aeration in
a medium composed of dehydrated Bacto-nutrient broth (0.8%),
glucose (1%), Na,HPO4 (0.6%), and KH2P04 (0.3%). When
maximum growth had occurred, the cells were harvested in a
1This publication was made possible through the support of
the Leukemia Society, Inc.
Received for publication March 31, 19(i(i.
refrigerated centrifuge. All subsequent steps in the enzyme prepa
ration were performed in the cold. The packed cells were sus
pended in 10 volumes distilled water, and the bacterial suspen
sion was sonically disrupted with a Branson 20-kc Sonifier for
3 min. The sonicate was centrifuged for 10 min at 1500 X g,
and the sediment was resuspended in half the volume of water
and subjected again to oscillation and centrifugation. This was
repeated until no sediment remained after centrifugation for 10
min at 1500 X g. All supernatants were combined and were
centrifuged at 9000 X g for 30 min.
The supernatant was mixed with 0.05 volume of l M MnCl2
solution, and the resultant precipitate was removed by eentrifuging for 30 min at 9000 X g- Ammonium sulfate was then added
to the supernatant to 90% saturation while maintaining pH 7.0
by dropwise addition of ammonium hydroxide. The precipitate
was collected by centrifugation, dissolved in a minimal volume
of 0.2 M phosphate buffer pH 7.0, and dialyzed against water.
The dialysate containing about 17 gm protein and 2.4 X IO4
units L-asparaginase activity was passed through a 3- x 25-cm
diethylaminoethyl cellulose (DEAE-cellulose) column which had
been equilibrated with 0.05 M phosphate buffer, pH 7.0. After
adsorption of the enzyme solution, development was initiated
with 150 ml of the 0.05 M phosphate buffer. Gradient elution was
then employed by adding 250 ml of a solution which was 0.5 M
NaCl and 0.05 M phosphate at pH 7.0 to a mixing chamber con
taining 250 ml of the initial 0.05 M phosphate buffer. Develop
ment of the chromâtogramwas completed by increasing the XaCl
concentration to 1.0 Mby the gradient technic. The flow rate was
75 ml/hr, and 15-ml fractions were collected. Aliquots of effluent
fractions were assayed for asparaginase activity. The active frac
tions under each peak were combined and concentrated by adding
ammonium sulfate to 90%. saturation. After eentrifugation, the
protein residues were dissolved in a minimal volume of 0.2 M
phosphate buffer, pH 7.0, dialyzed against water, lyophilized, and
stored at -5°C.
ANIMALEXPERIMENTS.
Lymphoma 6C3HED-OG ascites cells
(9) carried in C3H/HEJ mice were used to evaluate the antitumor activity of asparaginase preparations. The tumor cell
line was kindly furnished by Dr. J. G. Kidd. The animals were
procured from the Jackson Laboratory, Bar Harbor. The weight
of the mice used in experiments was 20-25 gm. Transplantation
of the tumor was performed by i.p. injection of 0.5-ml aliquots
containing approximately IO7 cells. The cell numbers were de
termined with the aid of a Coulter electronic cell counter. On
Days 4, 6, and 8 subsequent to tumor implantation the mice were
treated by i.p. injection of 0.3 ml asparaginase solution.
L-ASPARAGINASE
ASSAY.Unless otherwise specified, L-asparagi
nase assays were performed on aliquots (0.5 ml or less) of sample
in a total volume of 2.0 ml containing 20 jumólesL-asparagine
OCTOBER 1906
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2213
Joseph Roberts, Morion D. Prager, and Nicholas Bachynsky
and 0.05 M Tris chloride buffer, pH 8.5. Incubations were con
ducted for 30 min at 37°C.The reaction was stopped by addition
of 0.1 ml of 1.5 M trichloracetic acid. The
was removed by centrifugation, and the
was determined by nesslerization. A unit
that amount of enzyme which will liberate
in 30 min at 37°C.
precipitated protein
liberated ammonia
of L-asparaginase is
1 /¿moleof ammonia
Results
FRACTIONATION
OF E. coli L-ASPAKAGiNASE.
Mashburn and
Wriston (8) showed that E. coli L-asparaginase inhibits tumors
which are sensitive to guinea pig serum. Results obtained in this
study indicate that the L-asparaginase activity of E. coli extracts
can be resolved into 2 components by chromatography on a
DEAE-cellulose column (Chart 1). All of the L-asparaginase
was eluted during the gradient to 0.5 M NaCl. The gradient to
1.0 M NaCl produced an additional protein peak but no further
amidase activity. About 75% of the eluted L-asparaginase ac
tivity appeared in the fractions under the 2nd peak.
ANTITUMOH
ACTIVITY
OF E.
COK L-ASPARAGINASE
COMPONENTS.
Representative studies of the antitumor activity of the 2 E.
coli L-asparaginase components are presented in Table 1. It was
found that 3 injections of 15 units (3.3 mg protein) each of Peak
I L-asparaginase brought about complete regression of lymphoma
6C3HED in C3H mice. The cured mice demonstrated active
immunity to subsequent tumor implants. In contrast 3 injections
of 15 units (1.8 mg protein) each of Peak II L-asparaginase had
no observable effect on the survival time of tumor-bearing ani
mals, and increasing the injected enzyme concentration 2-fold
was without effect on the survival time (Table 1).
SOME PROPERTIES OF THE E. Culi L-ASPARAGINASECOMPO
NENTS.An investigation was undertaken to determine properties
of the L-asparaginase of Peak I and Peak II which might account
for the difference in their antitumor activity. Some of the pre
liminary findings are presented in this report.
Experiment shows that Peak I L-asparaginase has optimum
activity at pH 7.5-8.6, and more than 90% of the maximum
amidase activity is retained between pH 6.3 and 7.5. In contrast
Peak II L-asparaginase has a pH optimum at about 8.5 and the
amidase activity drops sharply at a pH less than 8 (Chart 2).
A study of the stability of the 2 E. coli amidase components
2.or
1.5
>
I-
o '-O
<
O
tr
en
0.5 -
10
CHART 1. DEAE-cellulose chromatogram
Fractions 22-28 was designated Peak II.
2214
15
TUBE
of E. coli L-asparaginase.
20
25
30
35
40
NUMBER
Fractions 16-18 were pooled and designated
CANCER
Peak I; the pool of
RESEARCH
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VOL. 20
Antitumor Activity of E. coli L-Asparaginase
3r
revealed that Peak II L-asparaginase is rapidly inactivated at
37°C,pH 6.8 (pH of the peritoneal ascitic fluid). Results sum
marized in Chart 3 show that the amidase activity of the Peak
II component drops nearly 50% within the first 30 min of incuba
tion at 37°Cin phosphate buffer and then remains constant over
the next 3 hr. The loss of activity (66%) of the Peak II L-as
paraginase is even more pronounced when the incubation is
carried out in peritoneal fluid. A similar 3-hr incubation of Peak
TABLE 1
INHIBITORYEFFECT OF E. coli L-ASPARAGINASEPEAKS I AND II
ON LYMPHOMA(5C3HED IN MICE"
TreatmentSaline
- 2
iu
(A
of
(days)11-13Tumor
time
mice9S85Survival
<
(0.85%)Peak
fully
regressed*9-1512-14 at
unitsPeak
I enzyme, 15
unitsPeak
II enzyme, 15
o.
(A
II enzyme, 30 unitsXo.
" Three injections were administered, each containing 15 units
L-aspa ragi nase.
6 Mice that regressed tumor showed active immunity to sub
sequent tumor implants.
0
]
INCUBATION
2
TIME
3
. HR
CHAUT 3. Stability of E. coli Peak I and Peak II L-aaparaginase.
Incubations were performed at 37°C,pH 6.8; 0.1-ml aliquota of the
4r
incubation mixture were removed at the designated times and
assayed for L-asparaginase activity. Peak I enzyme, 0.05 M phos
phate buffer (•
•).Peak I enzyme, C3H mouse ascitic fluid
(O
O). Peak II enzyme, 0.05 M phosphate buffer (A
A).
Peak II enzyme, C3H mouse ascitic fluid (A
A).
</>
(—
TABLE 2
IMMUNOLOGICRESPONSE TO E. coli L-ASPAUAGINASE
IN C3H MICE»
z
>»—
TreatmentSaline
>
t—
O
(0.85%)
Peak I (15 units)
Peak I (15 units)"Survival
<
?
O
(days)10-12
time
Tumor fully regressed
9-13
" Five mice in each test group. Animals were treated on Days
4, 6, and 8 after tumor implantation.
b Prior immunization with 4 15-unit injections of Peak I L-as
paraginase at 7-day intervals. The last injection was adminis
tered 23 days before tumor implant.
2
<x.
o.
CO
7
8
10
11
pH
CHART 2. Effect of pH on E. coli L-asparaginase Peak I and
Peak II activity. Peak I (•
•);Peak II (O
O). Assays
were performed in a buffer that was 0.0285 Meach with respect to
citric acid, barbital, KH2PC>4 and boric acid. The pH was adjusted
to the desired value with 0.2 M NaOH. The pH was determined at
the start and completion of the enzymatic assay.
II L-asparaginase in mouse serum and 0.5 M phosphate buffer,
pH 7.3, yielded drops in enzyme activity of 51 and 36%, re
spectively. In contrast, the Peak I amidase was stable over
the 3-hr period of incubation under similar conditions. The
retention of partial activity in the Peak II component may be
due to some contamination by Peak I amidase, or it may repre
sent a stable degradation product of Peak II L-asparaginase with
partial activity.
More extensive purification and characterization of the E.
coli L-asparaginase of Peaks I and II is currently under way in
this laboratory. It appears that the inability of Peak II L-as-
OCTOBER 1900
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2215
Joseph Huberts, Morton D. Prager, and Nicholas Bachynsky
TABLE 3
INHIBITION OF E. coli PEAK I L-ASPAHAGINASEACTIVITY BY
IMMUNEC3H MOUSE SERUM"
have revealed a number of factors which may influence the
therapeutic usefulness of enzymes such as L-asparaginase.
Attempts to purify L-asparaginase from E. coli by DEAEcellulose
column chromatography
resulted in the separation of
L-asparaginase inL-aspara
the
enzyme
activity
hito
2
components.
The early emerging
L-asparaginaseControlSerum
Material incubated with
activity(units)3.53.51.82.4Decrease
ginaseactivity
component (Peak I L-asparaginase)
contained less than 25%
(%)04932
of the total recovered enzyme activity. The Peak I amidase,
a potent inhibitor of the 6C3HED lymphoma, maintained 90%
mice6Serum
from untreated
of its maximum activity over a broad pH range (6.3-8.6), and
micefSerum
from L-asparaginase treated
was stable on prolonged incubation in mouse serum or peritoneal
treatedwith
from tumor-hearing mice
ascitic fluid. In contrast Peak II L-asparaginase which lacked
L-asparaginaseResidual
antitumor activity exhibited a marked decline in activity at pH
below 8 and was significantly inactivated by mouse serum and
" Equal volumes of enzyme solution and sera were incubated
for 30 min at 37°C;0.1-ml aliquota of the incubation mixture were peritoneal ascitic fluid. While the difference in the pH optimum
and the stability of Peak I and Peak II enzymes may account
assayed for L-asparaginase activity.
in part for the difference in tumor-inhibitory
properties, it should
* Sera were peols from 5 mice.
c See Footnote 6 of Table 2 for details of immunization.
be noted that in the observations reported here there was always
significant residual Peak II activity. Thus, with higher doses
of
Peak II enzyme, some inhibition might be observable as was
paraginase to inhibit the 6C3HED asparagine-requiring tumor
the case for guinea pig liver asparaginase (10). Current investiga
may be due at least in part to its instability in mouse serum
tions are concerned with additional factors that may contribute
and peritoneal fluids. This amidase differs from the yeast enzyme,
to understanding the difference in antitumor activity.
since the latter has l>een shown to be stable to prolonged incu
The Peak II amidase differs from the yeast L-asparaginase
bation with C3H mouse serum (4). The yeast enzyme is believed
in that the latter is not inactivated by mouse serum. The lack
to be an ineffective tumor inhibitor because of its rapid rate of
of antitumor activity of the yeast enzyme has been attributed
clearance in C3H mice by cells of the reticuloendothelial system.
to rapid uptake by cells of the reticuloendothelial
system (4).
IMMUNOLOGIC
RKSPONSK TO THE E. Culi L-ASPARAGINASE.
An
The reasons for the poor tumor-inhibitory
properties of the guinea
important consideration in the use of L-asparaginase as a tumorpig liver and B. coagulans L-asparaginase have not yet been de
inhibitory agent is the immunologie response of the recipient to
termined.
the enzyme. An antibody produced against the amidase as a
The E. coli Peak I L-asparaginase differs from the guinea pig
result of prolonged treatment could counteract the therapeutic
serum amidase in that the former is capable of eliciting an im
effectiveness of the enzyme. Results summarized in Table 2
mune response in C3H mice (Tables 2, 3). Broome (4) has re
show that when C3H mice are treated with L-asparaginase
ported that the guinea pig serum enzyme is of low antigenicity
before tumor implantation,
subsequent therapy with this en
in C3H mice. It seems, therefore, that the use of an enzyme
zyme proves ineffective. The antigenic properties of the enzyme
derived from an organism closely related genetically to the re
preparation were confirmed by a positive line of precipitation on
cipient would lessen the possibility of eliciting an immune re
a gel diffusion (Micro-EIek) slide employing mouse antiserum.
sponse in the host. Also treatment with large doses of enzyme over
A test of specific serum antibody against L-asparaginase was
a short period of time would appear to be highly desirable in
performed by incubating
a constant amount of serum with
tumor therapy.
increasing concentrations
of enzyme and then assaying for
asparaginase
activity. In several experiments, immune serum
Acknowledgments
consistently
decreased the amidase activity by up to 50%
The authors wish to thank Mr. Harvey Kincaid for his skilled
(Table 3). This finding is in agreement with that reported by
technical assistance in certain phases of this work.
El-Asmar and Greenberg (5) who performed a similar experiment
employing bacterial glutaminase and rabbit antiserum.
References
Since no more than half of the L-asparaginase activity was
1. Boyse, E. A., Old, L. .!., and Stockert. E. Inhibitory Effect of
inhibited by the immune serum, partial tumor inhibition and
(iuinea Pig Serum on a Number of New Leukemias in Mice.
increased survival time might have teen anticipated
in the
Nature, 198: 800, 19(13.
immunized animals, but this was not found to be the case. It
2. Broome, J. D. Evidence That the L-Asparaginase Activity of
may be that the complexing of enzyme with antibody leads to
(iuinea Pig Serum is Responsible for Its Antilymphoma Ef
an accelerated rate of destruction and disappearance of asparagi
fects. Ibid., 191: 1114-15, 19(51.
nase activity.
3. - —.Evidence that the L-Asparaginase of Guinea Pig Serum
is Responsible for its Antilymphoma Effects. I. Properties of
the L-Asparaginase of (iuinea Pig Serum in Relation to those
Discussion
of the Antilymphoma
Substance. J. Exptl. Med., ¡18:99-120,
Recent findings that L-asparaginase isolated from a variety
19G3.
of cells, e.g., yeast (4), B. coagulans (8), and guinea pig liver
4. — —. Antilymphoma
Activity of L-Asparaginase In Vivo.
(10), had little or no inhibitory effect against asparagine-requiring
Clearance Rates of Enzyme Preparations
from Guinea Pig
tumors warrant a more critical investigation of the function of
Serum and Yeast in Relation to their Effect on Tumor Growth.
J. Nati. Cancer Inst., 35: 967-74, 1965.
this enzyme in tumor therapy. Results obtained in this study
2216
CANCER
RESEARCH
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VOL. 20
Antitumor Activity of E. coli i-Asparaginase
5. El-Asmar, F. A., and Greenberg, D. M. Studies on the Mecha
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98: 505-82, 1953.
8. Mashburn, L. T., and Wriston, J. C., Jr. Tumor Inhibitory
OCTOBER
Effect of i,-Asparaginase from Escherichia coli. Arch. Biochem.
Biophys., 106: 451-52, 1964.
9. Sobin, L. H., and Kidd, J. G. A Metabolic Difference Between
Two Lines of Lymphoma OC3HED Cells in Relation to As
paragine. Proc. Soc. Exptl. Biol. Med., 119: 325-27, 19C5.
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11. Yellin, T. O., and Wriston, J. C., Jr. Antagonism of Purified
Asparginase
from Guinea Pig Serum toward Lymphoma.
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190(i
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2217
The Antitumor Activity of Escherichia coli l-Asparaginase
Joseph Roberts, Morton D. Prager and Nicholas Bachynsky
Cancer Res 1966;26:2213-2217.
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