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Isolation and characterization of four toxic protein fractions from the sea anemone Anemonia sulcata
To:loop, 1975, Vol. 13, DP . 353-337. Parpmon Prep. Printed in drat Btitsln.
ISOLATION AND CHARACTERIZATION OF FOUR TOXIC PROTEIN FRACTIONS FROM THE SEA ANEMONE ANEMONIA SULCATA L. C~lt>E1.LO and A. D'Aral>~.l.o Department of Physiology and Biochemistry, Stazione Zoologica, Naples, Italy (Acceptedforpublication 16 Apr11197~ L. CnxiBU.o and A. D'Ata>EU.o. Isolation and characterization of four toxic protein fractions from the seaanemone Anemonia sulcata. Toxicon l3, 353-357,1975 . A simple and rapidmethod is describedforthe isolationfrom the sea anemoneAnemonia sincere of four protein fractions of low molecular weight which have lethal activity against crabs. These proteins have been partially purified on ion exchange resins Dowex 50 W and CM cellulose and have been examined by venal electrophoresis and micro iscelectric focusing . The stability of the fractions was determined under various experimental conditions . The fractions have strong paralytic action on Carcinus mamas. The toxic activity, however, greatly differs from one fraction to another. INTRODUCTION
the pioneering studies by R1c1mT and Po1tTI>;tt (1936) it has become evident that most of the active compounds identified in Cnidaria are not responsible for its lethal effects (see WELSH et al., 1961, for refereaoes). Substances of low molecular weight such as tertiary amines and quaternary bases may function as non-specific pain producers or facilitate the absorption of the toxic components . The compounds responsible for death appear to be polypeptides or proteins of low molecular weight . The pharmacologically active substances and the toxic proteins are located in the cnidoblast (LANE, 1960; PHILLIPS, 1956); their presence in the whole body and tentacle tissue, however, cannot be excluded . Attempts to isolate toxic proteins from Anemonia sincere have been reported by B>htPSs and Blues (1971) who isolated two polypoptides, of molecular weight in the range 500010,000, having a strong paralysing effect on crabs and mammals. More recently, Noveiz et al. (1973), by precipitation with ammonium sulfate and chromatography, obtained from the tentacles of the same species a single protein fraction of a molecular weight of about 6000 daltons as determined by gel filtration . The protein, electrophoretically not homogeneous, was toxic to rats when injected intravenously . In this paper a simple and rapid method is described, by which four toxic protein components have been obtained from the whole tissues of Anemonia sincere . It is not known, however, if these toxic components are all or in part localized in the nematocysts. Some chemical and physiological properties of the proteins are also reported . SINCE
MATERIALS AND METHODS Living specimens of Anemonia sulcato were collected from the Bay of Naples and were kept in the aquarium with circulating wasted sea water until use. Batches of 1 kg were macerated in 1 1. of 0~ 1 N HCl solution for 12 hr at 4°C; the material was then oentrifu®ed at 9000 g for 10 min. The sediment was ra extracted and centrifuged as before until the supernatant showed no lethal action upon crabs. The com353 TOXICON 1973 Vol. 13
354
L. CARIELLO and A. D'ANIELLO
biped extracts were dialysed at 4`C against water for 24 hr with frequent changes. Cold acetone was addul slowly to a concentration of SO ~. The precipitate was discarded by filtration and the liquid was concentrated to about 1 1. under reduced pressure at room temperature. The crude concentrated extract was passed through a column of Dowex 50 W (100-200 meth, 2~ crosslinkage, H+ form) ; after washing with 0~5 N HCl and then with water, the material was eluted with 0~5 N NH,, dialysed against distilled water and lyophilized. The lyophilized material was redissolved in a small volume of water and chromatographed on a CM-cellulose column (2~3 x 22 cm) previously equilibrated with 005 M Na, HPO,-0025 M citric acid buffer, pH 4~8. Elution was carried out using a linear gradient to pH 7~8 and increasing molar concentrations of the buffer up to 0~1 M. The flow rate was 26 ml per hr. Fraction I was further purified by passing through a Sephadex G-50 column (105 x 65 cm) equilibrated with 0~2 M NaCI at a Bow rate of 20 ml per hr . All fractions were desalted in a Sephadex G-10 column (1 ~5 x 85 cm) and then lyophilized . Electrophoresis was carried out on Gelman cellulose polyacetate strips in 005 M phosphate-citrate buffer pH 4 at 300 V for 90 min . The bands were stained with 0~2 ~ amido black in methanol-acetic acidwater (4 :4 : 1 v/v) and washed with anhydrous methanol . Micro iscelectric focusing was performed according to the method of ('ATS~nIrooLAS (1968) . The starting current was S mA per gel rnlumn at 150 V for 1 hr. The molecular weight of the isolated fractions wasestimated on Biogel P-10 ; vo was determined with blue dextran 200 (Pharmacia, Uppsala) . FAD, trypsin inhibitor (Sigma) and Cyt. c were used as standards. Biogel P-10 columns (1 ~05 x 31 cm) were eluted with 0~2 M NaCI at a flow rate of 12 ml per hr. The effect of acetylcholinesterase was tested according to the method described by AaDON and Moxns (1965) using a purified enzyme preparation purchased from Sigma. Apyrase activity was tested with the method described by MEISTER (1947) using ATP and ADP from Sigma. The action on red blood cells was investigated by adding 015 ml of saline containing 17 ug of protein to S ml of a suspension of human erythrocytes in isotonic Johnson Lindsay buffer pH 7~2. After 1 hr the samples were centrifuged and the hemoglobin level in the supernatant was determined spectrophotometrically . The lethality was estimated by injecting intraperitoneally increasing quantities of the protein fractions (from 1 to 40 ug dissolved in sea water) into samples of Carclnus mamas (50 individuals for each dose). The crabs were paralysed in a time ranging from 13 to 20 sec. The lethal dose was taken as the quantity at which all animals died . RESULTS AND DISCUSSION
13y passing the extract through a CM-cellulose column, four fractions toxic for crabs are obtained (Fig. 1). The toxicity increased after purification on CM-cellulose ; no further
7000
~ooo é 000 a P E ~i000 u
2n
zooo 1000
Elution volume, mt
FRACTIONATION OF THE CRUDE EXTRACT ON CM-CELLULOSE . The sample wntained 342 mg of protein. The hatched areas indicate the tonic fractions. The continuous line represents pH values . FIG . 1 .
TOXICON 1973 Vol. l3
Tonic Protein Fractions from the Sea Anemone
35 5
increase, however, was obtained when the fractions were passed through Sephadex G-50 (Table 1). As revealed by electrophoresis, each fraction contains more than one component : TARIE 1 . PURIFICATION OF THE
Crude toxin Toxins after Dowex SO W Toxins after CM~ellulose Toxin l after Sephadex G-50
I II III IV
TOXnVS
Volume (ml) 1071 90 -
FROM
Arumonia aukata
Protein (mg) 3000 342 164 SS 17 10 76
Activity (CU) 605 5882 5269 1791 1575 6451 5714
Total activity 1815 x 10' 2012 x 10' 8641 x 10' 9850 x 10' 265 x 10' 645 x 10' 4342 x 10'
Activity is expressed in arbitrary units CU (Carcinus Unit), defined as the inverse of the amount of protein in mg which paralysed all crabs (20 g body weight) in an average time of S min.
three in the first fraction and two in the others (Fig. 2). Fractions I and II have the same electrophoresis behaviour as the toxins isolated by B~it>rss and B~Riss (1971) from the same animal . A
/ so"~ ®ZO"ti
/io "a 6 5 "~
~11II110 00011 a I 110 00011 z II 11 >z I 11
>II
Iu 1
1
FIG. 2. ELECI1tOPHO1tEST .4 ON GELMAN CELLULO~ POLYACETATE. A, Crude extract; B. Partiallypurified extract ; I : Fractions after Sephadex G-50 ; II, III and IV Fractions after CM-cellulose .
The degree of purity of each fraction, as determined by micro iscelectric focusing, is about 90~. Values from 410 to 585 were found for the iscelectric point (Fig. 3). As measured by gel filtration, the molecular weight of the isolated fractions ranges from 4400 to 5600 (Fig . 4). Each fraction is lethal when injected into crabs, the approximate Lnloo doses (lIg per kg crab] being 100, 100, 600 and 2~5 for fractions 1-4, respectively. They retain their lethality when kept in an acidic (0~2 N HCl) or in a basic (O~S N NaOH) medium. The lethality is also retained after heating at 70°C for 10 min in a neutral medium but is completely lost at low or high pH values. None of the fractions showed acetylcholinesterase or ATPase activity, nor were these two enzymes inhibited by the toxins ; no hemolysis was observed on human erythrocytes. The polypeptides isolated from Anemonia sulcata have been reported to have a strong paralysing action on the rabbit (B~It>;ss and B>`ItHSS, 1971) and to show cardiotropic effects on the rat (NovAx et al., 1973). Our fractions, when injected intravenously in the mouse, TOXICON 1973 Vol. 13
356
L. CARIELLO and A. D'ANIELLO
a Ec a ai
N
0
c0 a a a2o ô a
â aio
o~io ~so a~oo
4~30
5~00
5~50
pH
6~00
8~60
7-00
T~SO
Z
B00
FIG . 3. I~IiCRO ISOELECI'RIC FOCUSING OF PURIFIED EXTRACTS USING AN AMPHOLITE WTIH A RANGe OF 3'00-10'OO .
pH
One ml gel was collected in each tube. The hatched area indicate the toxic fractions.
lop mw
FIG. 4. MOLECULAR WEIGHT OF THE TOXIC FRACTIONS AS DETERS>IriED ON BIOGEL P-LO .
produced a transient motor inhibition and some rapidly reversible effects on the heart. Work is in progress for a better characterization of their pharmacological properties . Acknowledgements-The authors are indebted to Professor F.
GHnurrn for advice and encoulageallent.
REFERENCES N. O. and Moxws, B. (1965) In : Methods of Enzymatic Analysts, p. 765, (BERGEMYER, H. U., Ed .). New York : Academic Press. BffitFSS, L. and BfrRESS, R. (1971) Reinigung zweier krabben lähmender toxin~ aus der sx aasmono Aximonia sulcata. Kieler Metrtsforsch. 27, 117. CATSIINPOOLA3, N. (1968) Micro isoelectric focusing in polyacrylamide gel columns. Analyt. Biothun. 26, 480 AHDON,
710XICON 1973 Vol. 1 J
Toxic Protein Fractions from the Sea Anemone
35 7
L~uvE, C. E. (1960) The toxin of Physalia nematocysts . Ann. N.Y. Acod. Sci. 90, 742. MetsrEtt, A. (1947) Adenosintriphosphatase activity of human serum. Science 106,167 . Nov~,tc, V., SxEZ, D., CAUKAR, G. and LÉSEZ, D. (1973) Partial purification of a toxin from tentacles of the sea anemone Anemonia sulcata. Toxicon 11, 411 . PHIf_LIP3, J. H. (1956) Isolation of the active nematocysts of Metrldium senile and their chemical composition . Nature, Load. 178, 932. Rtct~r, C. and Pott~mt, P. (1936) Recherches surla toxine des Ccelentérés et les phénomènes d'anaphylaxie. Résult. Comp acimt. Prince Albert I95, 3. WBtstt, J. H. (1961) In : The Biology of Hydra, (LENHOFF, H. M. and LooMts, W. F., Eds.). Miami : University of Miami Pies .
nvncoH r~rs vd. t~
ISOLATION AND CHARACTERIZATION OF FOUR TOXIC PROTEIN FRACTIONS FROM THE SEA ANEMONE ANEMONIA SULCATA L. C~lt>E1.LO and A. D'Aral>~.l.o Department of Physiology and Biochemistry, Stazione Zoologica, Naples, Italy (Acceptedforpublication 16 Apr11197~ L. CnxiBU.o and A. D'Ata>EU.o. Isolation and characterization of four toxic protein fractions from the seaanemone Anemonia sulcata. Toxicon l3, 353-357,1975 . A simple and rapidmethod is describedforthe isolationfrom the sea anemoneAnemonia sincere of four protein fractions of low molecular weight which have lethal activity against crabs. These proteins have been partially purified on ion exchange resins Dowex 50 W and CM cellulose and have been examined by venal electrophoresis and micro iscelectric focusing . The stability of the fractions was determined under various experimental conditions . The fractions have strong paralytic action on Carcinus mamas. The toxic activity, however, greatly differs from one fraction to another. INTRODUCTION
the pioneering studies by R1c1mT and Po1tTI>;tt (1936) it has become evident that most of the active compounds identified in Cnidaria are not responsible for its lethal effects (see WELSH et al., 1961, for refereaoes). Substances of low molecular weight such as tertiary amines and quaternary bases may function as non-specific pain producers or facilitate the absorption of the toxic components . The compounds responsible for death appear to be polypeptides or proteins of low molecular weight . The pharmacologically active substances and the toxic proteins are located in the cnidoblast (LANE, 1960; PHILLIPS, 1956); their presence in the whole body and tentacle tissue, however, cannot be excluded . Attempts to isolate toxic proteins from Anemonia sincere have been reported by B>htPSs and Blues (1971) who isolated two polypoptides, of molecular weight in the range 500010,000, having a strong paralysing effect on crabs and mammals. More recently, Noveiz et al. (1973), by precipitation with ammonium sulfate and chromatography, obtained from the tentacles of the same species a single protein fraction of a molecular weight of about 6000 daltons as determined by gel filtration . The protein, electrophoretically not homogeneous, was toxic to rats when injected intravenously . In this paper a simple and rapid method is described, by which four toxic protein components have been obtained from the whole tissues of Anemonia sincere . It is not known, however, if these toxic components are all or in part localized in the nematocysts. Some chemical and physiological properties of the proteins are also reported . SINCE
MATERIALS AND METHODS Living specimens of Anemonia sulcato were collected from the Bay of Naples and were kept in the aquarium with circulating wasted sea water until use. Batches of 1 kg were macerated in 1 1. of 0~ 1 N HCl solution for 12 hr at 4°C; the material was then oentrifu®ed at 9000 g for 10 min. The sediment was ra extracted and centrifuged as before until the supernatant showed no lethal action upon crabs. The com353 TOXICON 1973 Vol. 13
354
L. CARIELLO and A. D'ANIELLO
biped extracts were dialysed at 4`C against water for 24 hr with frequent changes. Cold acetone was addul slowly to a concentration of SO ~. The precipitate was discarded by filtration and the liquid was concentrated to about 1 1. under reduced pressure at room temperature. The crude concentrated extract was passed through a column of Dowex 50 W (100-200 meth, 2~ crosslinkage, H+ form) ; after washing with 0~5 N HCl and then with water, the material was eluted with 0~5 N NH,, dialysed against distilled water and lyophilized. The lyophilized material was redissolved in a small volume of water and chromatographed on a CM-cellulose column (2~3 x 22 cm) previously equilibrated with 005 M Na, HPO,-0025 M citric acid buffer, pH 4~8. Elution was carried out using a linear gradient to pH 7~8 and increasing molar concentrations of the buffer up to 0~1 M. The flow rate was 26 ml per hr. Fraction I was further purified by passing through a Sephadex G-50 column (105 x 65 cm) equilibrated with 0~2 M NaCI at a Bow rate of 20 ml per hr . All fractions were desalted in a Sephadex G-10 column (1 ~5 x 85 cm) and then lyophilized . Electrophoresis was carried out on Gelman cellulose polyacetate strips in 005 M phosphate-citrate buffer pH 4 at 300 V for 90 min . The bands were stained with 0~2 ~ amido black in methanol-acetic acidwater (4 :4 : 1 v/v) and washed with anhydrous methanol . Micro iscelectric focusing was performed according to the method of ('ATS~nIrooLAS (1968) . The starting current was S mA per gel rnlumn at 150 V for 1 hr. The molecular weight of the isolated fractions wasestimated on Biogel P-10 ; vo was determined with blue dextran 200 (Pharmacia, Uppsala) . FAD, trypsin inhibitor (Sigma) and Cyt. c were used as standards. Biogel P-10 columns (1 ~05 x 31 cm) were eluted with 0~2 M NaCI at a flow rate of 12 ml per hr. The effect of acetylcholinesterase was tested according to the method described by AaDON and Moxns (1965) using a purified enzyme preparation purchased from Sigma. Apyrase activity was tested with the method described by MEISTER (1947) using ATP and ADP from Sigma. The action on red blood cells was investigated by adding 015 ml of saline containing 17 ug of protein to S ml of a suspension of human erythrocytes in isotonic Johnson Lindsay buffer pH 7~2. After 1 hr the samples were centrifuged and the hemoglobin level in the supernatant was determined spectrophotometrically . The lethality was estimated by injecting intraperitoneally increasing quantities of the protein fractions (from 1 to 40 ug dissolved in sea water) into samples of Carclnus mamas (50 individuals for each dose). The crabs were paralysed in a time ranging from 13 to 20 sec. The lethal dose was taken as the quantity at which all animals died . RESULTS AND DISCUSSION
13y passing the extract through a CM-cellulose column, four fractions toxic for crabs are obtained (Fig. 1). The toxicity increased after purification on CM-cellulose ; no further
7000
~ooo é 000 a P E ~i000 u
2n
zooo 1000
Elution volume, mt
FRACTIONATION OF THE CRUDE EXTRACT ON CM-CELLULOSE . The sample wntained 342 mg of protein. The hatched areas indicate the tonic fractions. The continuous line represents pH values . FIG . 1 .
TOXICON 1973 Vol. l3
Tonic Protein Fractions from the Sea Anemone
35 5
increase, however, was obtained when the fractions were passed through Sephadex G-50 (Table 1). As revealed by electrophoresis, each fraction contains more than one component : TARIE 1 . PURIFICATION OF THE
Crude toxin Toxins after Dowex SO W Toxins after CM~ellulose Toxin l after Sephadex G-50
I II III IV
TOXnVS
Volume (ml) 1071 90 -
FROM
Arumonia aukata
Protein (mg) 3000 342 164 SS 17 10 76
Activity (CU) 605 5882 5269 1791 1575 6451 5714
Total activity 1815 x 10' 2012 x 10' 8641 x 10' 9850 x 10' 265 x 10' 645 x 10' 4342 x 10'
Activity is expressed in arbitrary units CU (Carcinus Unit), defined as the inverse of the amount of protein in mg which paralysed all crabs (20 g body weight) in an average time of S min.
three in the first fraction and two in the others (Fig. 2). Fractions I and II have the same electrophoresis behaviour as the toxins isolated by B~it>rss and B~Riss (1971) from the same animal . A
/ so"~ ®ZO"ti
/io "a 6 5 "~
~11II110 00011 a I 110 00011 z II 11 >z I 11
>II
Iu 1
1
FIG. 2. ELECI1tOPHO1tEST .4 ON GELMAN CELLULO~ POLYACETATE. A, Crude extract; B. Partiallypurified extract ; I : Fractions after Sephadex G-50 ; II, III and IV Fractions after CM-cellulose .
The degree of purity of each fraction, as determined by micro iscelectric focusing, is about 90~. Values from 410 to 585 were found for the iscelectric point (Fig. 3). As measured by gel filtration, the molecular weight of the isolated fractions ranges from 4400 to 5600 (Fig . 4). Each fraction is lethal when injected into crabs, the approximate Lnloo doses (lIg per kg crab] being 100, 100, 600 and 2~5 for fractions 1-4, respectively. They retain their lethality when kept in an acidic (0~2 N HCl) or in a basic (O~S N NaOH) medium. The lethality is also retained after heating at 70°C for 10 min in a neutral medium but is completely lost at low or high pH values. None of the fractions showed acetylcholinesterase or ATPase activity, nor were these two enzymes inhibited by the toxins ; no hemolysis was observed on human erythrocytes. The polypeptides isolated from Anemonia sulcata have been reported to have a strong paralysing action on the rabbit (B~It>;ss and B>`ItHSS, 1971) and to show cardiotropic effects on the rat (NovAx et al., 1973). Our fractions, when injected intravenously in the mouse, TOXICON 1973 Vol. 13
356
L. CARIELLO and A. D'ANIELLO
a Ec a ai
N
0
c0 a a a2o ô a
â aio
o~io ~so a~oo
4~30
5~00
5~50
pH
6~00
8~60
7-00
T~SO
Z
B00
FIG . 3. I~IiCRO ISOELECI'RIC FOCUSING OF PURIFIED EXTRACTS USING AN AMPHOLITE WTIH A RANGe OF 3'00-10'OO .
pH
One ml gel was collected in each tube. The hatched area indicate the toxic fractions.
lop mw
FIG. 4. MOLECULAR WEIGHT OF THE TOXIC FRACTIONS AS DETERS>IriED ON BIOGEL P-LO .
produced a transient motor inhibition and some rapidly reversible effects on the heart. Work is in progress for a better characterization of their pharmacological properties . Acknowledgements-The authors are indebted to Professor F.
GHnurrn for advice and encoulageallent.
REFERENCES N. O. and Moxws, B. (1965) In : Methods of Enzymatic Analysts, p. 765, (BERGEMYER, H. U., Ed .). New York : Academic Press. BffitFSS, L. and BfrRESS, R. (1971) Reinigung zweier krabben lähmender toxin~ aus der sx aasmono Aximonia sulcata. Kieler Metrtsforsch. 27, 117. CATSIINPOOLA3, N. (1968) Micro isoelectric focusing in polyacrylamide gel columns. Analyt. Biothun. 26, 480 AHDON,
710XICON 1973 Vol. 1 J
Toxic Protein Fractions from the Sea Anemone
35 7
L~uvE, C. E. (1960) The toxin of Physalia nematocysts . Ann. N.Y. Acod. Sci. 90, 742. MetsrEtt, A. (1947) Adenosintriphosphatase activity of human serum. Science 106,167 . Nov~,tc, V., SxEZ, D., CAUKAR, G. and LÉSEZ, D. (1973) Partial purification of a toxin from tentacles of the sea anemone Anemonia sulcata. Toxicon 11, 411 . PHIf_LIP3, J. H. (1956) Isolation of the active nematocysts of Metrldium senile and their chemical composition . Nature, Load. 178, 932. Rtct~r, C. and Pott~mt, P. (1936) Recherches surla toxine des Ccelentérés et les phénomènes d'anaphylaxie. Résult. Comp acimt. Prince Albert I95, 3. WBtstt, J. H. (1961) In : The Biology of Hydra, (LENHOFF, H. M. and LooMts, W. F., Eds.). Miami : University of Miami Pies .
nvncoH r~rs vd. t~