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Isolation of eburnetoxin, A vasoactive substance from the Conus eburneus venom
Life Sciences, Vol. 31, pp. 1085-1091 Printed in the U;S.A.
ISOLATION OF EBURNETOXIN,
Pergamon Press
A VASOACTIVE SUBSTANCE FROM THE CONUS EBURNEUS VENOM
Jun'ichi Kobayashi, Hideshi Nakamura, Yoshimasa Hirata* and Yasushi Ohizumi Mitsubishi-Kasei Institute of Life Sciences, ii Minamiooya, Machida-shi, Tokyo 194 *Faculty of Pharmacy, Meijo Univeristy, Tempaku, Nagoya 468, Japan (Received in final form June 25, 1982) Summary Eburnetoxin, a powerful vasoactive protein has been isolated from the venom of the marine snail Conu8 eburneus, monitored by the contractile effect to the rabbit aorta. The molecular weight was estimated to be 28,000 by gel permeation chromatography and slab gel electrophoresis. The purified protein was electrophoretically homogeneous. The toxin at concentrations above 3 × i0 -? g/ml elicited a marked contractile response of aorta, which was inhibited by verapamll (10 -6 M). The minimum lethal dose in the fish Rhodeus ocellatus smithi was i ~g/g body weight. Most species belonging to the gastropod family Conidae possess potent venoms which are used primarily in the capture of prey. Kohn (I) has shown that the various species of Conidae appear to be pisclvorous, molluscivorous or vermivorous. It has been suggested that Conu8 eburneu8 is vermivorous, although the venom is toxic to blennies and gastropods as well as polychaete worms (2). The chemical and pharmacological properties of the venom have not been examined at all. In our preliminary study on the pharmacological action of the venoms of Conidae (3,4,5), the venom of C. eburneu8 has been found to cause a marked long-lasting contraction of the isolated rabbit aorta, which is inhibited by treatment with verapamil. In the present work, the isolation of eburnetoxin (ETX), a potent vasoactlve component from the C. eburneus venom, is described. Materials and Methods Enzymes and Chemicals. Trypsin was purchased from Sigma Chemical Co. and achymotrypsln was the product of Worthington Biochemical Corp. Phosphorylase b, bovine serum albumin, ovalbumin, chymotrypsinogen A, carbonic anhydrase, trypsin inhibitor and =-lactalbumin were purchased from Pharmacia Fine Chemicals as gel filtration and electrophoresis calibration kits. Verapamil hydrochloride was obtained from Eizai Co. Acrylamide was purchased from Selkagaku Kogyo Co. Sodium dodecyl sulfate (SDS) was obtained from Merck. Coomassle brilliant blue R-250 was purchased from Nakarai Chemicals Co. Blue Dextran 2000 was obtained from Pharmacla. Other chemicals were of chemical grade. Bioassay method. Rabbits (2-3 Kg) were sacrificed by cervical dislocation. The procedure of preparing the aorta and the technique for measurement of the contractile response were carried out as described previously (5). The contracting activity was estimated from the amount of protein which is required 0024-3205/82/111085-07503.00/0 Copyright (c) 1982 Pergamon Press Ltd.
1086
Eburnetoxin from Conu8 eburneus
Vol. 31, No. ii, 1982
to cause 50% of the maximal response (EDs0) of the aorta. The activity was expressed in unit per mg of protein. Protein was determined by the method of Lowry (6) using bovine serum albumin as the standard protein. Slab s e l electrophoresis. SDS/polyacrylamide slab gel electrophoresis was performed as described by Laemmli (7). The resolving and spacer gels contained 12 and 3% acrylamide, respectively. Electrophoresis was carried out at 60 mA for 4 hr. Proteins were stained by incubating the slab gel for 1 hr at 37°C in 0.1% Coomassie brilliant blue freshly dissolved in 25% isopropanol - 10% acetic acid and destained by a diffusion method in 10% acetic acid. Crude venom. Specimens of Conu8 eburneus Brugui~re were obtained from reefs in Okinawa waters. The gastropods were i~Inediately frozen, shipped by air to Tokyo and stored at -200C until the specimens were used. The shell of each specimen was cracked and the venom duct was dissected out of the animals after thawing. Venom was stripped from the excised venom ducts, weighed and suspended in 20 mM phosphate buffer (pH 7.0) at a concentration of 10 -1 g/ml. After the suspension was centrifuged (i0,000 g x i0 min), the supernatant was decanted and combined. Affinity chromatography. The extract of crude venom was loaded on a BlueSepharose CL-6B column (1.6 x 15 cm) equilibrated with 20 mM phosphate buffer (pH 7.0) at 4°C. After adequate washing, 0.5 M NaCI in the same buffer was introduced. Aliquots of 8 ml were monitored at 280 nm (Shimadzu Spectrophotometer UV-210A) and tested for the vasoconstricting activity. Active fractions were combined. Dialysis. The active fractions following affinity chromatography were dialyzed in seamless cellulose tubing. The dialysis was performed in the 20 mM phosphate buffer (pH 7.0) at 4°C for 12 hr. The retentate in the tube was collected and their absorbance at 280 nm and vasoconstricting activities were measured. Isoeleetric focusin$ column chromatosraphy. The active fraction after dialysis was applied to an isoelectric focusing column (40 ml capacity) with 0-40% sucrose density gradient containing 1% carrier ampholine (LKB Ampholine, pH 3.5-9.5). After focusing at 400 V for 48 hr at 4°C, the solution in the column was fractionated. Absorbance at 280 nm, pH and vasoconstricting activities of aliquots of 0.6 ml were measured, respectively. High performance liquid chromatography. High performance liquid chromatography (HPLC) of proteins was carried out using two TSK gel G 3000 SW columns (7.5 mm x 30 m m, Toyo Soda Co.) in series with 20 mM phosphate buffer (pH 7.0) containing 0.2 M NaCI. An Altex Model 100A solvent delivery system was used to pump solvents through the columns at flow rate of 1 ml/min. Samples of 1 to 40 Dg in volumes of 5 to 20 ~i were injected with a Water Model U6K injector. The column effluent was monitored at 280 nm with a JASCO UVIDEC-100 III variable wavelength detector and tested for vasoconstricting activity. Enzymatic digestion. The effects of incubating ETX in 0.2 M Tris-HCl buffer (pH 7.5) for 12 hr at 37°C with trypsin and a-chymotrypsin were examined. After incubation, the extracts were tested for activity on the aorta. Lethal activity in fish. Fishes Rhodeus ocellatus smithi (Regan) weighing 3-4 g were injected intraperitoneally (i.p.) with ETX. The minimum lethal dose was taken at 24-hr period.
Vol. 31, No. ii, 1982
Eburnetoxin from Conus eburneus
1087
Results Isolation of ETX. Vasoconstricting activity was eluted with 0.5 M NaC1 during the affinity chromatography of the crude venom of Conus e b ~ e u s on a BlueSepharose CL-6B column (Fig. i). One major peak appeared in HPLC of the active fractions by gel permeation chromatography on G 3000 SW columns, whereas two neighboring major bands of proteins were detected in SDS/polyacrylamide slab gel electrophoresis of the active fractions (Fig. 2c). As shown in Table i, the first fractionation caused 22 times increase of the vasoconstricting activity.
1.0 T
0.8
e-
&
v
E '- 0.6
:~IXIO
> . m
(M
o 0.4
2.000
%% %%%
u
-B 0.2
,000
.¢
0
20
c . m
40 60 80 Volume (ml)
100
4"J
tO u 0
0
FIG. i Affinity chromatography of the crude venom of Conus e b ~ e u s on a Blue Sepharose CL-6B column. After washing with 20 mM phosphate buffer, pH 7.0 (not written), the column was eluted with buffered 0.5 M NaCI.
TABLE i Vasoconstricting activity of eburnetoxin (ETX) during purification on the rabbit isolated aorta. ED50 a)
Activity b)
(~g protein/ml)
(units/mg)
ii.0
91
ETX after affinity chromatography
0.5
2,000
ETX after electrofocusing
0.2
5,000
Crude venom
a) The minimum concentration of protein required for arise in 50% of the maximal contraction in the aorta. b) Vasoconstricting activity was expressed in unit per mg of protein.
1088
Eburnetoxln from Conu8 ebur~¢u8
Q
b
c
Vol. 31, No. 11, 1982
d
D
Q D
D
TX
W !
D
FIG. 2 Analysis of eburnetoxln (ETX) by 12% SDS/polyacrylamlde slab gel eleetrophoresls. (a) Standard proteins: phosphorylase b (Mr 94,000), bovine serum albumin (Mr 67,000), ovalbumin (Mr 43,000), carbonic anhydrase (Mr 30,000), trypsin inhibitor (Mr 20,100) and ~-lactalbumin (Mr 14,400), (b) crude venom of Conu8 eburneus, (c) ETX after affinity chromatography and (d) ETX after electrofocuslng.
I
pi=4.8
0.4
,-'"
<
1
E 0.3
tO
182
°j..
50.2
0.1 ,o~.°
.j ,
20
40 60 Fraction No.
80
FIG. 3 Electrofocusing chromatography on a column with 0-40% sucrose density gradient containing 1% carrier ampholine (LKB, pH 3.5-9.5). After focusing at 400V for 48 hr at 4°C, fractions of 0.6 ml were collected.
Eburnetoxln from Conu8 eburneu8
Vol. 31, No. II, 1982
1089
This active fraction was further purified by ampholine electrofocusing column chromatography with sucrose density gradient. The active component was eluted as a single peak during electrofocusing (Fig. 3). The purified component, ETX, gave a single band of protein in slab gel electrophoresis (Fig. 2d) and one peak in HPLC on G 3000 SW column (Fig. 4B). Properties of ETX. The molecular weight of ETX was estimated to be 28,000 by SDS/polyacrylamide slab gel electrophoresis (Fig. 2) and gel permeation chromatography on G 3000 SW columns (Fig. 5). ETX following electrophoresis on acrylamide gel was stained by 0.1% Coomassie brilliant blue. The vasoconstricting activity appeared around pH 4.8 during electrofocusing. Samples of ETX in 20 mM phosphate buffer (pH 7.0) maintained at 25°C for i day showed 60% loss in activity compared with that of control samples when tested on the aorta. ETX lost completely the activity by incubation with either the proteases, trypsin or ~-chymotrypsin. The activity of ETX was also lost by boiling at pH 7.0 for 15 min or by treatment with i N NaOH or i N HCI at 25°C for i day. Vasoconstrictin$ activity on.the aorta. ETX at concentrations above 3 x 10 -7 g/ml elicited a marked long-lasting contraction of the rabbit aorta. The contractile response to ETX was inhibited by treatment with verapamil (10 -6 M) (Fig. 6). Lethal actlvity in fish. was i ~g/g body weight.
The minimum lethal dose of ETX in the fish Rhodeu8
B
A d
0OO4 I:I
0
,<
•
|
•
|
•
.
0
|
.
.
8
a
.
|
12
time(rain) FIG. 4 Chromatography on two TSK gel G 3000 SW columns in series. (A) Standard: (a) Blue Dextran, (b) bovine serum albumin, (c) ovalbumin and (d) chymotrypsinogen A. (B) Eburnetoxin (ETX). They were eluted from the columns with 20 mM phosphate buffer (pH 7.0) containing 0.2 M NaCI at a flow rate of i ml/min.
1090
Eburnetoxln from Conus eburneus
Vol. 31, No. ii, 1982
2.2 motryrsinogen A
2.0
(28,0OO) -,, 1.8 1.6
Albumin "e
\
1./.
log mol.wt. FIG. 5 Molecular weight determination of eburnetoxin (ETX) by gel filtration on G 3000 SW columns using chymotrypsinogen A (Mr 25,000), ovalbumin (Mr 43,000) and bovine serum albumin (Mr 67,000) as standard proteins. k' represents retention relative to void volume (Vo) Vo was determined with Blue Dextran.
lOmin _
Ilg
_
f ETX
f
Verap
ETX FIG. 6
Effect of eburnetoxin (ETX, 3 x 10 -7 g/ml) on the rabbit isolated aorta in the presence or absence of verapamil (Verap, 10 -6 M). Verap was added 15 min before administration of ETX.
Vol. 31, No.
II, 1982
Eburnetoxin
from Conus e b u ~ e u s
1091
Discussion In our preliminary study (3), the venom of Conus e b ~ e u s has been found to cause a marked contraction of the rabbit aorta, which is abolished by verapamil. In the present work, the active principle in the venom has been purified by affinity chromatography and subsequent electrofocusing, monitored by the vasoconstricting activity on the aorta. The active component isolated from the venom showed a single band during slab gel electrophoresis and a single peak in HPLC on gel. The activity of the component was completely lost hy incubation with proteases and by boiling. These results strongly suggest that the active material is a protein. The vasoactive substance was named eburnetoxin. The molecular weight of ETX was estimated as 28,000 by gel electrophoresis and high performance gel filtration. The isoelectric point (pl) of ETX was considered to be 4.8. ETX causes a marked contraction of the aorta at concentrations above 3 × 10 -7 g/ml. The minimum effective dose of ETX is estimated to be 8 × 10 -9 M, considered as mol. wt. 28,000 of ETX. The contraction induced by ETX was inhibited by a Ca-antagonist (verapamil), suggesting that ETX increases the verapamil sensitive Ca L+ influx across smooth muscle membrane of the aorta, resulting in contractions. The natural food of C. e b ~ e u s remains unknown, although the gastropod has been tentatively classified as a worm-eater Conidae from consideration of the morphological features of the venom apparatus (2). The venoms of most worm-eater Conidae are toxic to worm but not to fish, whereas those of some vermivorous Conidae involving C. e b ~ e u s are toxic to fish as well as worm (2). ETX isolated from the C. e b ~ e u s venom must be a major component in the venom toxic to fish, since the minimum lethal dose of ETX in the fish Rhodeus is very small (i ~g/g body weight) and even comparable to that of the active principle in the venom of a fish-eater Conidae (8).
Acknowledgement The authors are grateful to Mr. Z. Nagahama of Okinawa for providing the specimens of C. eb~r~eus and to Miss. R. Abe of the present institute for her skillful technical assistance.
References i. 2. 3. 4. 5. 6. 7. 8.
A.J. KOHN, Ecol. Monogr. 29 47-90 (1959). R. ENDEAN and C. RUDKIN, Toxicon 2 225-249 (1965). J. KOBAYASHI, H. NAKAMURA, Y. HIRATA and Y. OHIZUMI, Toxicon, in press. J. KOBAYASHI, H. NAKAMURA and Y. OHIZUMI, Br. J. Pharmac. 73 583-585 (198~. J. KOBAYASHI, Y. OHIZUMI, H. NAKAMURA and Y. HIRATA, Toxicon 19 757-762 (1981). O.H. LOWRY, N.J. ROSEBROUGH, A.L. FARR and R.J. RANDALL, J. Biol. Chem. 193 265-275 (1951). U.K. LAEMMLI, Nature (London) 227 680-685 (1970). J. KOBAYASHI, H. NAKAMURA, Y. HIRATA and Y. OHIZUMI, Biochem. Biophys. Res. Com~un. 105 1389-1395 (1982).
ISOLATION OF EBURNETOXIN,
Pergamon Press
A VASOACTIVE SUBSTANCE FROM THE CONUS EBURNEUS VENOM
Jun'ichi Kobayashi, Hideshi Nakamura, Yoshimasa Hirata* and Yasushi Ohizumi Mitsubishi-Kasei Institute of Life Sciences, ii Minamiooya, Machida-shi, Tokyo 194 *Faculty of Pharmacy, Meijo Univeristy, Tempaku, Nagoya 468, Japan (Received in final form June 25, 1982) Summary Eburnetoxin, a powerful vasoactive protein has been isolated from the venom of the marine snail Conu8 eburneus, monitored by the contractile effect to the rabbit aorta. The molecular weight was estimated to be 28,000 by gel permeation chromatography and slab gel electrophoresis. The purified protein was electrophoretically homogeneous. The toxin at concentrations above 3 × i0 -? g/ml elicited a marked contractile response of aorta, which was inhibited by verapamll (10 -6 M). The minimum lethal dose in the fish Rhodeus ocellatus smithi was i ~g/g body weight. Most species belonging to the gastropod family Conidae possess potent venoms which are used primarily in the capture of prey. Kohn (I) has shown that the various species of Conidae appear to be pisclvorous, molluscivorous or vermivorous. It has been suggested that Conu8 eburneu8 is vermivorous, although the venom is toxic to blennies and gastropods as well as polychaete worms (2). The chemical and pharmacological properties of the venom have not been examined at all. In our preliminary study on the pharmacological action of the venoms of Conidae (3,4,5), the venom of C. eburneu8 has been found to cause a marked long-lasting contraction of the isolated rabbit aorta, which is inhibited by treatment with verapamil. In the present work, the isolation of eburnetoxin (ETX), a potent vasoactlve component from the C. eburneus venom, is described. Materials and Methods Enzymes and Chemicals. Trypsin was purchased from Sigma Chemical Co. and achymotrypsln was the product of Worthington Biochemical Corp. Phosphorylase b, bovine serum albumin, ovalbumin, chymotrypsinogen A, carbonic anhydrase, trypsin inhibitor and =-lactalbumin were purchased from Pharmacia Fine Chemicals as gel filtration and electrophoresis calibration kits. Verapamil hydrochloride was obtained from Eizai Co. Acrylamide was purchased from Selkagaku Kogyo Co. Sodium dodecyl sulfate (SDS) was obtained from Merck. Coomassle brilliant blue R-250 was purchased from Nakarai Chemicals Co. Blue Dextran 2000 was obtained from Pharmacla. Other chemicals were of chemical grade. Bioassay method. Rabbits (2-3 Kg) were sacrificed by cervical dislocation. The procedure of preparing the aorta and the technique for measurement of the contractile response were carried out as described previously (5). The contracting activity was estimated from the amount of protein which is required 0024-3205/82/111085-07503.00/0 Copyright (c) 1982 Pergamon Press Ltd.
1086
Eburnetoxin from Conu8 eburneus
Vol. 31, No. ii, 1982
to cause 50% of the maximal response (EDs0) of the aorta. The activity was expressed in unit per mg of protein. Protein was determined by the method of Lowry (6) using bovine serum albumin as the standard protein. Slab s e l electrophoresis. SDS/polyacrylamide slab gel electrophoresis was performed as described by Laemmli (7). The resolving and spacer gels contained 12 and 3% acrylamide, respectively. Electrophoresis was carried out at 60 mA for 4 hr. Proteins were stained by incubating the slab gel for 1 hr at 37°C in 0.1% Coomassie brilliant blue freshly dissolved in 25% isopropanol - 10% acetic acid and destained by a diffusion method in 10% acetic acid. Crude venom. Specimens of Conu8 eburneus Brugui~re were obtained from reefs in Okinawa waters. The gastropods were i~Inediately frozen, shipped by air to Tokyo and stored at -200C until the specimens were used. The shell of each specimen was cracked and the venom duct was dissected out of the animals after thawing. Venom was stripped from the excised venom ducts, weighed and suspended in 20 mM phosphate buffer (pH 7.0) at a concentration of 10 -1 g/ml. After the suspension was centrifuged (i0,000 g x i0 min), the supernatant was decanted and combined. Affinity chromatography. The extract of crude venom was loaded on a BlueSepharose CL-6B column (1.6 x 15 cm) equilibrated with 20 mM phosphate buffer (pH 7.0) at 4°C. After adequate washing, 0.5 M NaCI in the same buffer was introduced. Aliquots of 8 ml were monitored at 280 nm (Shimadzu Spectrophotometer UV-210A) and tested for the vasoconstricting activity. Active fractions were combined. Dialysis. The active fractions following affinity chromatography were dialyzed in seamless cellulose tubing. The dialysis was performed in the 20 mM phosphate buffer (pH 7.0) at 4°C for 12 hr. The retentate in the tube was collected and their absorbance at 280 nm and vasoconstricting activities were measured. Isoeleetric focusin$ column chromatosraphy. The active fraction after dialysis was applied to an isoelectric focusing column (40 ml capacity) with 0-40% sucrose density gradient containing 1% carrier ampholine (LKB Ampholine, pH 3.5-9.5). After focusing at 400 V for 48 hr at 4°C, the solution in the column was fractionated. Absorbance at 280 nm, pH and vasoconstricting activities of aliquots of 0.6 ml were measured, respectively. High performance liquid chromatography. High performance liquid chromatography (HPLC) of proteins was carried out using two TSK gel G 3000 SW columns (7.5 mm x 30 m m, Toyo Soda Co.) in series with 20 mM phosphate buffer (pH 7.0) containing 0.2 M NaCI. An Altex Model 100A solvent delivery system was used to pump solvents through the columns at flow rate of 1 ml/min. Samples of 1 to 40 Dg in volumes of 5 to 20 ~i were injected with a Water Model U6K injector. The column effluent was monitored at 280 nm with a JASCO UVIDEC-100 III variable wavelength detector and tested for vasoconstricting activity. Enzymatic digestion. The effects of incubating ETX in 0.2 M Tris-HCl buffer (pH 7.5) for 12 hr at 37°C with trypsin and a-chymotrypsin were examined. After incubation, the extracts were tested for activity on the aorta. Lethal activity in fish. Fishes Rhodeus ocellatus smithi (Regan) weighing 3-4 g were injected intraperitoneally (i.p.) with ETX. The minimum lethal dose was taken at 24-hr period.
Vol. 31, No. ii, 1982
Eburnetoxin from Conus eburneus
1087
Results Isolation of ETX. Vasoconstricting activity was eluted with 0.5 M NaC1 during the affinity chromatography of the crude venom of Conus e b ~ e u s on a BlueSepharose CL-6B column (Fig. i). One major peak appeared in HPLC of the active fractions by gel permeation chromatography on G 3000 SW columns, whereas two neighboring major bands of proteins were detected in SDS/polyacrylamide slab gel electrophoresis of the active fractions (Fig. 2c). As shown in Table i, the first fractionation caused 22 times increase of the vasoconstricting activity.
1.0 T
0.8
e-
&
v
E '- 0.6
:~IXIO
> . m
(M
o 0.4
2.000
%% %%%
u
-B 0.2
,000
.¢
0
20
c . m
40 60 80 Volume (ml)
100
4"J
tO u 0
0
FIG. i Affinity chromatography of the crude venom of Conus e b ~ e u s on a Blue Sepharose CL-6B column. After washing with 20 mM phosphate buffer, pH 7.0 (not written), the column was eluted with buffered 0.5 M NaCI.
TABLE i Vasoconstricting activity of eburnetoxin (ETX) during purification on the rabbit isolated aorta. ED50 a)
Activity b)
(~g protein/ml)
(units/mg)
ii.0
91
ETX after affinity chromatography
0.5
2,000
ETX after electrofocusing
0.2
5,000
Crude venom
a) The minimum concentration of protein required for arise in 50% of the maximal contraction in the aorta. b) Vasoconstricting activity was expressed in unit per mg of protein.
1088
Eburnetoxln from Conu8 ebur~¢u8
Q
b
c
Vol. 31, No. 11, 1982
d
D
Q D
D
TX
W !
D
FIG. 2 Analysis of eburnetoxln (ETX) by 12% SDS/polyacrylamlde slab gel eleetrophoresls. (a) Standard proteins: phosphorylase b (Mr 94,000), bovine serum albumin (Mr 67,000), ovalbumin (Mr 43,000), carbonic anhydrase (Mr 30,000), trypsin inhibitor (Mr 20,100) and ~-lactalbumin (Mr 14,400), (b) crude venom of Conu8 eburneus, (c) ETX after affinity chromatography and (d) ETX after electrofocuslng.
I
pi=4.8
0.4
,-'"
<
1
E 0.3
tO
182
°j..
50.2
0.1 ,o~.°
.j ,
20
40 60 Fraction No.
80
FIG. 3 Electrofocusing chromatography on a column with 0-40% sucrose density gradient containing 1% carrier ampholine (LKB, pH 3.5-9.5). After focusing at 400V for 48 hr at 4°C, fractions of 0.6 ml were collected.
Eburnetoxln from Conu8 eburneu8
Vol. 31, No. II, 1982
1089
This active fraction was further purified by ampholine electrofocusing column chromatography with sucrose density gradient. The active component was eluted as a single peak during electrofocusing (Fig. 3). The purified component, ETX, gave a single band of protein in slab gel electrophoresis (Fig. 2d) and one peak in HPLC on G 3000 SW column (Fig. 4B). Properties of ETX. The molecular weight of ETX was estimated to be 28,000 by SDS/polyacrylamide slab gel electrophoresis (Fig. 2) and gel permeation chromatography on G 3000 SW columns (Fig. 5). ETX following electrophoresis on acrylamide gel was stained by 0.1% Coomassie brilliant blue. The vasoconstricting activity appeared around pH 4.8 during electrofocusing. Samples of ETX in 20 mM phosphate buffer (pH 7.0) maintained at 25°C for i day showed 60% loss in activity compared with that of control samples when tested on the aorta. ETX lost completely the activity by incubation with either the proteases, trypsin or ~-chymotrypsin. The activity of ETX was also lost by boiling at pH 7.0 for 15 min or by treatment with i N NaOH or i N HCI at 25°C for i day. Vasoconstrictin$ activity on.the aorta. ETX at concentrations above 3 x 10 -7 g/ml elicited a marked long-lasting contraction of the rabbit aorta. The contractile response to ETX was inhibited by treatment with verapamil (10 -6 M) (Fig. 6). Lethal actlvity in fish. was i ~g/g body weight.
The minimum lethal dose of ETX in the fish Rhodeu8
B
A d
0OO4 I:I
0
,<
•
|
•
|
•
.
0
|
.
.
8
a
.
|
12
time(rain) FIG. 4 Chromatography on two TSK gel G 3000 SW columns in series. (A) Standard: (a) Blue Dextran, (b) bovine serum albumin, (c) ovalbumin and (d) chymotrypsinogen A. (B) Eburnetoxin (ETX). They were eluted from the columns with 20 mM phosphate buffer (pH 7.0) containing 0.2 M NaCI at a flow rate of i ml/min.
1090
Eburnetoxln from Conus eburneus
Vol. 31, No. ii, 1982
2.2 motryrsinogen A
2.0
(28,0OO) -,, 1.8 1.6
Albumin "e
\
1./.
log mol.wt. FIG. 5 Molecular weight determination of eburnetoxin (ETX) by gel filtration on G 3000 SW columns using chymotrypsinogen A (Mr 25,000), ovalbumin (Mr 43,000) and bovine serum albumin (Mr 67,000) as standard proteins. k' represents retention relative to void volume (Vo) Vo was determined with Blue Dextran.
lOmin _
Ilg
_
f ETX
f
Verap
ETX FIG. 6
Effect of eburnetoxin (ETX, 3 x 10 -7 g/ml) on the rabbit isolated aorta in the presence or absence of verapamil (Verap, 10 -6 M). Verap was added 15 min before administration of ETX.
Vol. 31, No.
II, 1982
Eburnetoxin
from Conus e b u ~ e u s
1091
Discussion In our preliminary study (3), the venom of Conus e b ~ e u s has been found to cause a marked contraction of the rabbit aorta, which is abolished by verapamil. In the present work, the active principle in the venom has been purified by affinity chromatography and subsequent electrofocusing, monitored by the vasoconstricting activity on the aorta. The active component isolated from the venom showed a single band during slab gel electrophoresis and a single peak in HPLC on gel. The activity of the component was completely lost hy incubation with proteases and by boiling. These results strongly suggest that the active material is a protein. The vasoactive substance was named eburnetoxin. The molecular weight of ETX was estimated as 28,000 by gel electrophoresis and high performance gel filtration. The isoelectric point (pl) of ETX was considered to be 4.8. ETX causes a marked contraction of the aorta at concentrations above 3 × 10 -7 g/ml. The minimum effective dose of ETX is estimated to be 8 × 10 -9 M, considered as mol. wt. 28,000 of ETX. The contraction induced by ETX was inhibited by a Ca-antagonist (verapamil), suggesting that ETX increases the verapamil sensitive Ca L+ influx across smooth muscle membrane of the aorta, resulting in contractions. The natural food of C. e b ~ e u s remains unknown, although the gastropod has been tentatively classified as a worm-eater Conidae from consideration of the morphological features of the venom apparatus (2). The venoms of most worm-eater Conidae are toxic to worm but not to fish, whereas those of some vermivorous Conidae involving C. e b ~ e u s are toxic to fish as well as worm (2). ETX isolated from the C. e b ~ e u s venom must be a major component in the venom toxic to fish, since the minimum lethal dose of ETX in the fish Rhodeus is very small (i ~g/g body weight) and even comparable to that of the active principle in the venom of a fish-eater Conidae (8).
Acknowledgement The authors are grateful to Mr. Z. Nagahama of Okinawa for providing the specimens of C. eb~r~eus and to Miss. R. Abe of the present institute for her skillful technical assistance.
References i. 2. 3. 4. 5. 6. 7. 8.
A.J. KOHN, Ecol. Monogr. 29 47-90 (1959). R. ENDEAN and C. RUDKIN, Toxicon 2 225-249 (1965). J. KOBAYASHI, H. NAKAMURA, Y. HIRATA and Y. OHIZUMI, Toxicon, in press. J. KOBAYASHI, H. NAKAMURA and Y. OHIZUMI, Br. J. Pharmac. 73 583-585 (198~. J. KOBAYASHI, Y. OHIZUMI, H. NAKAMURA and Y. HIRATA, Toxicon 19 757-762 (1981). O.H. LOWRY, N.J. ROSEBROUGH, A.L. FARR and R.J. RANDALL, J. Biol. Chem. 193 265-275 (1951). U.K. LAEMMLI, Nature (London) 227 680-685 (1970). J. KOBAYASHI, H. NAKAMURA, Y. HIRATA and Y. OHIZUMI, Biochem. Biophys. Res. Com~un. 105 1389-1395 (1982).