[CANCER RESEARCH 49, 3834-3838, July 15, 1989]
Isolation and Characterization
Sea Hare, Aplysia kurodai1
of a Novel Cytolytic Factor in Purple Fluid of the
Masatoshi Yamazaki, Katsuya Kinuira, Jun Kisugi, Koji Muramoto, and Hisao Kumiya
Faculty of Pharmaceutical Sciences, Teikyo University, Sagamiko-cho, Tsukui-gun, Kanagawa 199-01 [M. Y., K. K., J. K.J, and School of Fisheries Sciences, Kitasato
University, Sanriku, Iwate 022-01, Japan fK. M., H. K.]
ABSTRACT
A novel cytolytic factor, aplysianin P, which induces tumor lysis, was
purified to apparent homogeneity from the purple fluid of the sea hare
Aplysia kurodai. Purified aplysianin P was a single M, 60,000 polypeptide. This factor was half-maximally active at 3-25 ng protein/ml and
lysed all the tumor cells tested but did not lyse normal WBC or RBC.
Aplysianin P was labile on treatments with heat, low pH, urea, and
periodate, but not with Promise. The factor completely inhibited the
syntheses of DNA, RNA, and protein by tumor cells within 2 h and
caused their complete cytolysis within 18 h. Tumor lysis by aplysianin P
was inhibited by ¿Y-acetylneuraminicacid, suggesting that recognition of
the sugar moiety is a key step in the cytolysis induced by aplysianin P.
The factor also prolonged the survival of mice bearing syngeneic MM46
ascites. It did not resemble previously isolated antineoplastic glycoproteins from the eggs (aplysianin E) or albumen gland (aplysianin A) of A.
kurodai in terms of molecular size, antigenicity, or amino acid composi
tion. These results suggest that aplysianin P found in an invertebrate, the
sea hare, is a new antitumor factor.
INTRODUCTION
Sea hares are opisthobranch mollusks which have attracted
the interest of many scientists investigating biologically active
substances (1-4). Most of these substances are low molecular
weight compounds derived from algal diets. We previously
recognized the presence of glycoprotein with antibacterial and
antineoplastic activity in eggs and albumen glands of Aplysia
kurodai, but not in other tissues (5, 6). The main active sub
stances were isolated as a M, 250,000 glycoprotein from eggs
(7) and a M, 320,000 glycoprotein from the albumen gland (8).
Most Aplysia species discharge a purple fluid from a purple
gland when they are disturbed. The nature of the purple dye in
the purple fluid of sea hares was elucidated by Rüdiger(9), but
no bioactive substance has yet been isolated from this fluid.
Here, we report the isolation and characterization of a novel
cytolytic factor (aplysianin P) from the purple fluid of A.
kurodai and compare some of the properties of aplysianin P
with cytolytic factors in eggs (aplysianin E) and albumen gland
(aplysianin A).
MATERIALS
AND METHODS
Collection of Purple Fluid of A. kurodai. Specimens of A. kurodai
were collected in Lake Humana. Shizuoka, Japan, in May and June.
The purple fluid was obtained by agitating the animals and was then
frozen at -80°C until use.
Purification Procedure. The purple fluid was dialyzed against 10 HIM
phosphate buffer and loaded onto columns (2.9 x S3 cm; 4 parallel
columns of DE52; Whatman, Maidstone, Kent, England) previously
equilibrated with starting buffer (10 HIM phosphate, pH 7.4). The
columns were washed with the starting buffer and then the retained
material was eluted with a gradient of 0.2-1 M NaCl. Fractions with
Received 9/15/88; revised 1/27/89; accepted 4/5/89.
The costs of publication of this article were defrayed in part by the payment
of page charges. This article must therefore be hereby marked advertisement in
accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
1This work was supported in part by a Grant-in-Aid for Cancer Research from
Kureha Chemical Industry' Co., Ltd. (Tokyo).
cytolytic activity were pooled, concentrated on a Toyo UK-10 mem
brane, and applied to a column (2.7 x 120 cm) of Sepharose 6B. Active
fractions eluted with phosphate-buffered saline were pooled, concen
trated as before, and applied to a column (1.9 x 105 cm) of Sephacryl
S-200. Fractions with cytolytic activity were dialyzed against 10 mM
phosphate buffer and loaded onto a column (1.0 x 1.3 cm) of DE52
previously equilibrated with 10 mM phosphate buffer, pH 7.4. The
cytolytic factor was eluted with a linear gradient of 0-0.4 M NaCl.
Mice and Tumor Cells. MM46, MM48, and MH134 tumor cells
were collected from the peritoneal cavity of C3H/He mice. 1.1210 and
P388 leukemic cells were collected from ascites of male DBA/2 mice.
EL 4 cells were obtained from the peritoneal cavity of C57BL/6 mice.
Human leukemic cells, Raji, Molt-3, and K-562, were harvested from
i/i vitro cultures. Cells of a human lung adenocarcinoma line, PC-6,
PC-7, and PC-9, which were kindly supplied by Dr. Saijo (National
Cancer Center, Tokyo, Japan) were also harvested from in vitro cultures.
Cytolytic Assay. Target tumor cells (2 x IO6 cells/ml) were labeled
with Na2"CrO4 (100 uCi/ml) in RPMI containing 10% fetal calf serum
for 2 h and then washed 3 times. Labeled target cells (5 x 10' cells)
with or without a test preparation in wells (7 mm in diameter) of flatbottomed microplates were incubated in 0.2 ml RPMI-fetal calf serum
for 18 h at 37°Cunder CO2 in air. The radioactivity of the supernatant
was measured and cytolytic activity was defined as
% of cytolysis =
Experimental count —¿
control count
Maximum releasable count —¿
control count x 100
Maximum release of 5'Cr was measured after freeze-thawing labeled
tumor cells 3 times. The control count was measured as the radioactivity
released spontaneously from labeled cells. Units of cytolytic activity
were calculated as
___ 50% cytolysis
Units = Final dilution giving
Neutralizing Test. Murine MM46 tumor cells were pretreated with
mitomycin C (a positive control) or aplysianin P in vitro at 37°Cfor 60
min. Then the cells (5 x IO4 cells/mouse) were inoculated i.p. into
C3H/He mice. The inoculum of 5 x IO2cells is the minimum number
of cells needed to obtain a tumor taken in this system.
Determination of Protein and Carbohydrate. Protein was measured by
the procedure of Lowry et al. (10) with bovine serum albumin as a
standard. Carbohydrate was determined by the method of Dubois et al.
(11) with glucose as a standard.
Assays of Synthetic Activities. The metabolic activities of tumor cells
with and without treatment with a cytolytic factor were measured in
terms of incorporation of tritiated thymidine, uridine, and leucine into
DNA, RNA, and protein, respectively. Samples of 1 x IO5 MM46
tumor cells were incubated with 1 ¿tCi
of [m<?/A>>/-1H]thymidine
(52 Ci/
mmol), 2 MCiof [5,6-3H]uridine (39 Ci/mmol), or 5 ¿iCiof L-[4,5-'H]
leucine (170 Ci/mmol; New England Nuclear, Boston, MA) in 0.2 ml
of RPMI-fetal calf serum at 37°Cfor 60 min. Then the cells were
harvested on filters and washed with Labo Mash LM-101. The filters
were dried and their radioactivity was counted in a liquid scintillation
spectrophotometer.
Polyacrylamide Gel Electrophoresis. SDS2-polyacrylamide gel electrophoresis was performed by the method of Laemmli (12) in 12.5%
acrylamide gels. Samples were heated at 100'C for 3 min in 1% SDS
in the presence of 2% 2-mercaptoethanol. The gels were stained with
! The abbreviation used is: SDS, sodium dodecyl sulfate.
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ANTITUMOR FACTOR FROM SEA HARE
Table 1 Purification of aplysianin P from purple fluid of a sea hare
Coomassie brilliant blue. Gradient gel electrophoresis was performed
in 4-30% polyacrylamide gel.
Various Treatments. pH stability was examined by adding l N HC1
or l N NaOH to 1-ml samples to give the desired pH values. The
samples were kept at 4°Cfor 30 min and then readjusted to pH 7.0
and dialyzed against phosphate-buffered saline, pH 7.4. Other 1-ml
samples were treated with 2-mercaptoethanol (0.1 M) or urea (8 M) at
37°Cfor 2 h and then dialyzed. Samples were treated with periodate in
the dark at 37°Cfor 6 h and then dialyzed. Samples of 1 mg were
incubated with 1 mg of Pronase (Calbiochem) at 30°Cfor 24 h.
Reagents. /V-Acetylneuraminic acid was purchased from Sigma.
Other monosaccharides were products of Wako Pure Chemical Co.
(Tokyo, Japan). Mitomycin C was purchased from Kyowa Hakko
Kogyo Co., Ltd. (Tokyo, Japan).
Amino Acid Analysis. Amino acid composition was analyzed by the
method of Chang et al. (13). Protein was hydrolyzed with 30 u\ of 6 M
HC1 at 110°(for 22 h in an evacuated tube and dried in a vacuum.
The hydrolysate was derivatized with 4-dimethylaminoazobenzene-4'-
cation(fold)114IIS216425Yield(
StepCrude
fluidDEAE-cellulose
ISepharose
6BSephacryl
S-200DEAE-cellulose
IIVolume(ml)800219.55.02.9Protein(mg)1,140767.63.71.6Sugar(mg)340241.70.80.35
100
50
sulfonyl chloride and diluted with 10 HIMsodium phosphate buffer (pH
6.5). The amino acid derivatives were separated on a Hypersil ODS
column (3 ¿im;4.6 x 100 mm). Amino acid sequence was analyzed as
described previously (14).
>>
_j
RESULTS
3
Purification of Aplysianin P from Purple Fluid. A cytolytic
factor (aplysianin P) was purified from the purple fluid (800 ml
from 100 No.) by two types of gel filtration and two cycles of
ion-exchange chromatography. The elution profiles on column
Chromatograph ics are shown in Fig. 1. The cytolytic activity
was eluted in a fraction of molecular weight of 50,000 with 260
mM NaCl. The purple dye was partly removed during the first
DE52 column chromatography and completely during Sepharose 6B column chromatography.
Table 1 summarizes the purification of aplysianin P. The
purified factor contained 18% sugar, suggesting that it was a
glycoprotein. This aplysianin P lysed target tumor cells at a
concentration of 5 ng protein/ml (Fig. 2). To confirm that the
purified factor was associated with cytolytic activity, we sub
jected it to electrophoresis on 4-30% acrylamide gel (Pharma
cia) and examined the cytolytic activity of slices of the gel. The
9
27
81
Concentration (no/ml)
Fig. 2. Dose dependence of the cytolytic effect of aplysianin P. "Cr-labeled
MM46 tumor cells were incubated with aplysianin P for 18 h. Cytolysis was
measured as the radioactivity of the supernatant. Points and bars, means ±SDfor
duplicate estimations.
A
B
C
D
E
F
G
94k
67k.
43k«
30k»
20k «
Fig. 3. SDS-polyacrylamide gel electrophoresis of purified aplysianin P. The
purified sample was subjected to electrophoresis on 12.5% acrylamide slab gel at
a constant voltage of 120 V for 3 h (A and G). Markers (phosphorylase B, M,
94.000; bovine serum albumin. M, 67,000; ovalbumin. M, 43,000: carbonic
anhydrase, M, 30.000; soybean trypsin inhibitor, M, 20,000; n-lactalbumin, M,
14,000). (B)
from (D)
dialysis
(50 from
pg protein).
from DEAE
cellulose
I (50Sample
«ig
protein).
Sample
Sepharose(C)6BSample
(10 Mgprotein).
(/•'(
Sample from Sephacryl S-200 (10 pg protein). (F) DEAE-cellulose II (10 ng
protein). A. thousands.
Fig. 1. Elution patterns of the cytolytic factor (aplysianin P) on column
chromatographies. The dialyzed purple fluid was applied to a DEAE-cellulose
column I (A) and the active fraction from this column was applied to Sepharose
6B (B). The active fraction from a Sepharose 6B column was applied to a
Sephacryl S300 column (C) and the active fraction from this column was purified
on DEAE-cellulose column II (D). Fractions (A, B, and C. 15 ml; D, l ml) were
tested for cytolytic activity (•),NaCl concentration (
), and absorbance at
280 nm (
). n-Chymotrypsinogen (M, 25,000). bovine serum albumin (M,
67,000), catalase (M, 232,000). and thyroglobulin (M, 669.000) were used as
marker proteins. A', molecular weight in thousands.
purified factor gave a single M, 60,000 band and the position
of cytolytic activity coincided with that of the protein band
(data not shown).
To examine the subunit structure of the cytolytic factor, we
subjected the purified aplysianin P to SDS-polyacrylamide gel
electrophoresis. Fig. 3 shows that the purified preparation gave
a single M, 60,000 protein band on SDS-polyacrylamide gel
electrophoresis. Treatment of the factor with 2-mercaptoetha
nol did not affect the molecular mass of the M, 60,000 com
ponent. These results indicate that the cytolytic factor, aplysi
anin P, is a M, 60,000 glycoprotein of a single polypeptide.
Characterization of Aplysianin P. The kinetics of cytolysis
was studied (Fig. 4). Tumor lysis by the cytolytic factor was
3835
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ANTITUMOR
FACTOR
FROM
SEA HARE
Table 3 Effects of various treatments on the stability ofaplysianin P
Residual
activity"
80
Treatment
None
pH 4.5 (4'C, 30 min)
pH 9.5 (4°C,30 min)
100
<5
50
Heat
55'C, 10 min
60°C,10 min
40
0
<5
8 M urea
<13
<6
0.01 M periodate
0.1 M 2-mercaptoethanol
100
Pronase (1 mg:l mg)
100
" Aplysianin P was treated as indicated. For details of treatments see "Materials
and Methods." Its cytolytic activity after treatment is expressed as residual activity
4
8
12
16
Incubation Tim«(hr)
as a percentage ofthat of the untreated preparation.
Fig. 4. Time course of tumor lysis by aplysianin P. "Cr-labeled MM46 tumor
cells were incubated with aplysianin P (0.5 ng protein/ml) for 18 h. Samples of
supernatant were taken at the times indicated for measurement of radioactivity.
Bars, SD.
100
Table 2 Target cell specificity ofaplysianin P
Target cells
Murine
MM46 (mammary carcinoma)
MH134 (hepatoma)
L12IO (leukemia)
P388 (leukemia)
YAC-1 (lymphoma)
EL-4 (lymphoma)
Human
K-562 (leukemia)
Raji (leukemia)
Molt-3 (leukemia)
PC-6 (lung adenocarcinoma)
PC-7 (lung adenocarcinoma)
PC-9 (lung adenocarcinoma)
Murine
Spleen cells
Blastogenic spleen cells
(ng/protein/ml)
o 3O
'S
10
8
3
5
*
II
4
0246
25
3
5
20
25
25
Incubation Time (hr)
Fig. 5. Effects of aplysianin P on syntheses of macromolecules in tumor cells.
MM46 tumor cells were incubated with or without aplysianin P (0.5 fig protein/
ml) and then with tritiated thymidine (O), uridine (•),or leucine (A) at 37°Cfor
60 min. The acid-insoluble fraction was obtained at the times indicated and its
radioactivity was measured.
>40.000
>40.000
% of control
=
cpm of factor-treated cells
cpm of untreated
Sheep
>40,000
Erythrocytes
" Target cells were incubated wtih aplysianin P for 18 h. Mi.,,, concentration
for 50% lysis of target cells.
cells
X 100
Points and bars, means ±SD for triplicate estimations.
% Cytolysis
50
time dependent and was complete within 18 h. Tumor lysis was
also examined by microscopy. After 8 h, the number of bubbles
in the target cells gradually increased, and after 10 h the cell
surface membrane burst (data not shown).
Next, we examined the cytolytic activity against various target
cells. As shown in Table 2, all the murine and human tumor
cells tested were lysed by purified aplysianin P in the concen
tration range of 3-25 ng protein/ml. In contrast, normal spleen
cells and erythrocytes were resistant to this cytolytic factor.
These results indicate that tumor cells are relatively susceptible
to aplysianin P.
Table 3 summarizes the sensitivity ofaplysianin P to various
treatments. Aplysianin P was heat labile, showing an apprecia
ble loss of activity after heating at 55°Cfor 10 min. The factor
was stable at neutral pH (6-8) but lost one-half of its activity
at pH 9.5 and all activity at pH 4.5. The cytolytic activity was
lost on treatment with 8 M urea or 0.01 M periodate, but not
on treatment with 0.1 M 2-mercaptoethanol or Pronase.
To determine the mechanism of cytolysis by aplysianin P, we
examined the correlation between cell metabolism and cytolysis.
Fig. 5 shows that the abilities of tumor cells to incorporate
thymidine, uridine, and leucine were completely inhibited by
the factor within 2 h. Thus, cytolysis may follow a decrease in
metabolic activities such as the synthesis of DNA, RNA, and
protein.
100
Control
D-Glucose
D-GalactoseD-Mannose
N-Acetyl
D-glucosamine
D-FucoseL-Fucose
/¿//////////////I'/////////////I//////////////I^ZZZZZZZZZZZZ31
/ / / /
D-RiboseD-ArabinoseL-Rhamnose
N-Acetyl
neuraminic acid'/////////////I////////////I/////////////I'/////////////I/////////////J'///////
1
Fig. 6. Effects of sugars on cytolysis by aplysianin P. "Cr-labeled MM46
tumor cells and 100 ng aplysianin P/ml were incubated at 37"C for 18 h with or
without the indicated sugars at a final concentration of 50 HIM.
To determine the mechanism of cell recognition by aplysianin
P, we examined the effects of various sugars on cytolysis. As
shown in Fig. 6, W-acetylneuraminic acid at a concentration of
50 HIM inhibited tumor lysis by aplysianin P, whereas nine
other sugars did not. Thus recognition of sialic acid may be an
initial step in the cytolysis induced by aplysianin P.
Table 4 shows that aplysianin P as well as mitomycin C had
a protective effect against the MM46 tumor. Some mice that
survived were resistant to challenge with a lethal dose of fresh
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ANTITUMOR
FACTOR FROM SEA HARE
Table 4 Neutralizing effects ofaplysianin P on MM46 tumor cells
Neutralization"TreatmentNone
days'10
mouse
Survival
ratio
Survival
ratio
days'0/5
aplysianin E. However, no precipitate was observed between
aplysianins P and E. Some properties of aplysianins are listed
in Table 6.
Survival
±0
±0.8221925
DISCUSSION
>45
Mitomycin C
Mg
4/52/34/518.2
10
units''
2 1.6 ±0.9 (20-24)
Aplysianin P
0/5
Sea hares, which are opisthobranch mollusks, have been
21.5 ±0.7
22)>45Challenge*Survival
(21.
Aplysianin P
40 units
3/55/518.0
Aplysianin PDose/
160 units0/55/5
reported to contain some biologically active substances, includ
" Murine MM46 tumor cells were pretreated with mitomycin C or aplysianin
ing antibacterial factors (1), toxins (3), and chemical defensive
P in vitro at 37°Cfor 60 min. Then the cells (5 x IO4cells/mouse) were inoculated
substances (2, 4). Most of these substances are low molecular
weight compounds derived from the algae on which the sea
hare feeds. However, no bioactive high molecular substances
except agglutinin have previously been identified in these ani
mals (15).
Here, we purified a novel cytolytic factor (aplysianin P) from
Table 5 Amino acid composition ofaplysianins P, E, and A
the purple fluid to a M, 60,000 homogeneous species. This
AminoacidAlaArgAsxCysGlxGlyHisIleLeuLysMetPheProSerThrTrpTyrVal°
factor is distinct from the purple dye previously reported (4)
%)10.53.46.80.87.111.80.64.08.22.62.43.612.95.910.7NT"2.26.6AplysianinE(mol
%)8.37.911.9013.79.01.83.77.55.21.64.04.76.93.2NT3.07.9AplysianinA"(mol
%)6.77.39.9010.38.13.92.88.28.91.54.24.17.04.7NT5.86.8
and is the first bioactive substance isolated from the purple
fluid of a sea hare. This factor seemed to be a glycoprotein of a
single polypeptide. Purified aplysianin P is labile on heat treat
ment or incubations at high and low pH values (Table 3), but
it was resistant to treatment with Pronase. There are several
possible explanations for this property: aplysianin P may have
a protease-resistant character (e.g., it may be a protease inhib
itor); proteases may liberate small peptides from it that have
cytolytic activity; or its active site for cytolysis may not be a
peptide. Further studies are needed on this point.
Aplysianin P inhibits the syntheses of macromolecules such
as DNA, RNA, and protein before causing cytolysis (Figs. 4
and 5). Furthermore, its cytolytic effect is temperature depend
ent (data not shown). These observations suggest that the
8.*
Data from Ref.
cytolytic process is closely related to cellular metabolism. In
NT, not tested.AplysianinP(mol
other words, aplysianin P does not act on target cells as a
detergent. Target cells may have a receptor for aplysianin P
since the cytolysis was inhibited by a monosaccharide (Fig. 6).
O <8>
Therefore, aplysianin P may have two characteristics conducive
to the killing of tumor cells: an ¡ml¡proliferativieffect, like an
anticancer drug; and the ability to selectively recognize target
cells.
Fig. 7. Ouchterlony double-diffusion test of aplysianins. Center well, rabbit
We isolated two antitumor factors, aplysianin E from eggs
anti-aplysianin E serum (10 fill. I. purified aplysianin P (80 nn nil. 10 j/ll: B,
(7) and aplysianin A from the albumin gland (8) of I. kurodai.
purified aplysianin E from eggs (95 Mg/ml, 10 /ill; C, purified aplysianin A from
Aplysianin E has a molecular weight of 250,000 and is com
albumin gland (58 »ig/ml,10 /ill
posed of three distinct subunits with molecular weights of
76,000, 88,000, and 102,000. Aplysianin A, which has a molec
tumor cells. Thus, aplysianin P had a protective effect against
ular
weight of approximately 320,000 and contains four A/r
MM46 ascites tumor.
85,000
subunits, is larger than aplysianin E. Moreover, these
Comparison of Properties of Aplysianin P with Properties of
two factors have a common antigen and similar amino acid
Aplysianins E and A. Examination of the amino acid composi
composition, suggesting that aplysianin A is a precursor of
tion of the aplysianin P revealed that it contained a relatively
large amount of proline, glycine, and threonine (Table 5). The aplysianin E. Aplysianin P, however, did not resemble these
sequence of the amino-terminal region of aplysianin P was
Table 6 Comparison of properties ofaplysianins P, E, and A
Thr-X-Gly-Pro-Ala-.
P
A
We previously isolated two cytolytic factors, aplysianin E
from purple
E
from albumen
from eggs of A. kurodai (7) and aplysianin A from the albumen
PropertyMolecular
fluid60,000118%260Pro,
eggs250,0003
from
gland320,0004
gland (8). Here, we examined the amino acid composition of wtSubunitSugar
x4)9.8%75Glx,
(M, 85,000
76,000,88,000,102,000)8%75Glx,
(M,
aplysianin P from purple fluid and compared it with the amino
acid compositions of aplysianins E and A. As shown in Table
5, the amino acid composition of aplysianin P was not similar
contentElution
NaCI
to those of aplysianins E or A, whereas the compositions of the (ITIM)Amino
acid composi
ThrrichNo
Gly,
richReaction~I8LabileAplysianin
Asx
richPartial
Asx
latter two were similar to each other.
tionReaction
to
anti-aplysi
To determine the antigenicity of aplysianin P, we examined
reaction-18LabileAplysianin
reaction(spur)~I8Labile
anin E serum (Ouch
whether it reacted to anti-aplysianin E antibody. As shown in
Fig. 7, Ouchterlony double-diffusion tests showed the spur of a terlony)Time
(h)Stability
for cytolysis
(55°C,10
to heat
cross-reaction between aplysianins E and A, suggesting that
min)Aplysianin
aplysianin A was partly identical in antigenic specificity to
i.p. into C3H/He mice. Data are representative of two similar experiments.
4 Fresh MM46 tumor cells (5 x IO4 cells/mouse) were inoculated i.p. into
surviving mice 50 days after neutralization.
' Mean ±SD.
'' One unit equals 0.26 *<gof protein.
3837
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ANTITUMOR
FACTOR FROM SEA HARE
two antineoplastic glycoproteins in terms of antigenicity or
amino acid composition, although the three factors have a
similar kinetics of cytolysis, a similar tumor spectrum and a
similar sensitivity to various treatments (Table 6). These results
suggest that aplysianin P is a different molecule from aplysianins A and E, although the structure responsible for cytolysis
may be similar among the three factors.
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Downloaded from cancerres.aacrjournals.org on June 16, 2017. © 1989 American Association for Cancer Research.
Isolation and Characterization of a Novel Cytolytic Factor in
Purple Fluid of the Sea Hare, Aplysia kurodai
Masatoshi Yamazaki, Katsuya Kimura, Jun Kisugi, et al.
Cancer Res 1989;49:3834-3838.
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Downloaded from cancerres.aacrjournals.org on June 16, 2017. © 1989 American Association for Cancer Research.