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Studies on abrin
4th International Symposium on Animal, Plant and Microbial Toxins
107
Lim, B.L.1 and SAwAi, Y.,' (1) Division of Medical Ecology, Institute for Medical Research, Kuala Lumpur, Malaysia and (2) The Japan Snake Institute, Yabuzuka Honmachi, Nittagun, Gunma, Japan. ECOLOGY AND DISTRIBUTION OF SEA SNAKES IN PENINSULAR MALAYSIA The sea snakes (Hydrophiinae) have a number of distinctive morphological features which include the reduction of complete degeneration of the ventral shields and a laterally flattened tail like the blade of an oar. All of them are venomous, having poison fangs situated anteriorly in the upper jaw. In Peninsular Malaysia,more than 20 species belonging to 12 genera are known to inhabit the Malaysian waters . TwEmm (1963) grouped these in two subfamilies, the Laticaudinae, with Laticauda colubrina and Aepyurus eydouxi, and Hydrophiinae, comprising all other species. With the exception of Hydrophis, all genera of sea snakes in local waters are represented by a single species. Sea snakes in Peninsular Malaysia are an occasional hazard to fishermen and bathers. A number of fatal cases of sea snake bite have been reported from Penang and other coastal towns (REm et al., 1963 ; Lnw and BAKAR,1970 ; SAwAi et al., 1971). Today very little is known of the ecology and distribution of this group of snakes . The snakes belonging to sub-family Laticaudinae are found in rocky islands and near the shore, hiding in crevices or under stones. Of the two species, Laticauda colubrina is the commonest, this is an amphibious snake, spending part of its time on drier places, particularly in crevices of rocks. Laticauda is the only genus of sea-snakes which is oviparous. Members of the Hydrophiinae can be divided into three different ecological stratifications. There are at least four species of sea snakes known, from the present study, to frequent river mouths, rocky shores, and mangrove shores, and five species are found to inhabit very deep waters usually miles away from the shores. The rest of the species have been known to inhabit deeper waters, but intermediate between the former two groups . It is often difficult for the victim to identify the sea snakes involved with the bite. The treatment of any snakebite depends greatly on the identification of the snake. Knowledge gained from the present study will give an indication of which species of the sea snakes arelikely to be involved depending on the type of areas that the victim visited at the time he was bitten, thus reflecting on the treatment of such cases. Lim, B. L. and DATo Asu BAKAR BIN InAmm (1970). Bites and stings by venomous animals with special reference to snake-bites in West Malaysia. Med. J. Malaya 25,1211-141 . Rte, H. A., TkmAN, P. C. and MARrna, W. T. (1963) Epidemiology of snake-bite in North Malaya . Br . med. J. 7, 992 . SAwAi, Y. and KOBA, K. (1971) Thevenomous snakes of Malaysia. 77re Snake 3,129-152. TwEEDrE, M. W. F. (1961) The Snakes of Malaya. Singapore: Government Printing Office .
LiN, J. Y., Institute of Biochemistry, College of Medicine, National Taiwan University, Tapei, Taiwan . STUDIES ON A NEW CARDIOTOXIN ISOLATED FROM THE EDIBLE MUSHROOM, VOLVARIELLA VOLVACEAE AND FLAMMULINA VELUTIPES Two new cardiotoxic proteins, volvatoxin andfiammutoxin have been isolated from Volvariella volvaceae and F7arnnwlina velutipes, respectively . The molecular weight of fiammutoxin was determined to be 11,000 while volvatoxin consisted of two non-identical subunits volvatoxin A1 and A s, with molecular weight of 23,000 and 50,000, respectively. The LDsu of flamrnutoxin was 2-5 mg per kg body weight and that of volvatoxin was 1-23 mg when the toxic proteins were administered by i.p . igjection into mice. In vitro, both toxic proteins caused the lyses of human erythrocyte in the absence of lecithin, and in vivo they induced EKG changes.
LiN, J. Y., Institute of Biochemistry, College of Medicine, National Taiwan University, Taipei, Taiwan . STUDIES ON ABRIN Abrin was subjected to aminoethylation with ethyleneimine and AE-abrin was fractionated with a CM52 Column Chromatography. Two subunits were obtained, and the molecular weight of the A and B subunit was determined to be 25,000 and 30,000, respectively . Both subunits have no biological activity as compared to native abrin. TO%ICON 1975 Vol. 13
108
4th International Symposium on Animal, Plant and Microbial Toxins
Lint, S . S ., HuANG, M ., TsENG, W. C . and LEE, C. Y., Pharmacological Institute, College of Medicine, National Taiwan University, Taipei, Taiwan. COMPARATIVE STUDIES ON THE MUSCULAR CONTRACTURE INDUCED BY COBRA CARDIOTO-NINN AND SCORPION TOXIN Both cobra cardiotoxin (CTX) and scorpion (Androctonus australis) toxin II are basic polypeptides containing four disulfide bonds ; their molecular weights are 6734 and 7249, respectively . Both toxins are potent in inducing contracture in chick biventer cervicis muscle preparation . Unlike CTX, however, toxin 11 can produce spontaneous contraction in this preparation . LTX contracture was completely abolished whereas toxin II contracture was not affected by high Call (10 mM) . The spontaneous contraction induced by toxin 11 was augmented increasing Ca'+ concentration from 2-5 to 5-0 mM . On the other hand, CTX but not toxin II contracture was accelerated by Cal l free medium . High Mg'+ (10 mM) accelerated CTX contracture but inhibited toxin 11 contracture . Lowering the extracellular Na+ from 143 to 83 mM was without effect on contracture induced by either toxin . Both high Mg'+ and low Na+ did not affect significantly the spontaneous contraction induced by toxin II . Both tetrodotoxin (0-3-3 uM) and procaine (1-8 mM) did not inhibit CT7{ contracture at all but inhibited toxin II contracture markedly. Toxin 11 contracture resumed as soon as these drugs were removed . Both D-tubocurarine (10 - ° g per ml) and ß-bungarotoxin (10 - ' g per ml) had no effect on either CTX or toxin II contracture, but they had a slight inhibitory effect on the spontaneous contraction induced by toxin II. GTX increased "Ca" uptake and caused the inward release of "Ca" in the chick biventer cervicis muscle. Studies on the effect of toxin 11 on "Call fluxes are in progress . The present results suggest that CTX induces contracture by releasing Call from the surface membrane of the muscle, while toxin II may have a different mechanism of action from CTX . As to the spontaneous contraction, toxin II may induce it by acting on the presynaptic region and the muscle membrane, since ß-bungarotoxin can only slightly inhibit it . (Scorpion toxin II was kindly supplied by Prof. F. Miranda, Lab, Biochem . Méd ., Fac . M6d., Marseille.) Lo, T.-B . and CFIANo, W.-C., Institute of Biological Chemistry, Academia Sinica and Institute of Biochemical Sciences, National Taiwan University, Taipei, Taiwan . STUDIES ON PHOSPHOLIPASE A FROM FORMOSAN COBRA (NAJA NAJA ATRA) VENOM Homogeneous phospholipase A from Formosan cobra (Naja naja atra) venom was isolated and purified by CM-cellulose, DEAF-cellulose column chromatography and Sephadex G-50 gel filtration . The specific activity was 400 jig per mg as assayed by titration method with synthetic ß, y-dipalrnito-L-a-lecithin as substrate. This enzyme showed high stability on storing and heating. Apparent Km and Vmax were determined at pH 7-5 and were found to be 9-0 x 10 - ' M and 0-5 lteq per min per ml, respectively . Maximal activity was exhibited at pH 7-1 and at pH 5-2 as measured by turbidimetric method with egg yolk suspension as substrate .Thetoxicity of highly purified phospholipase A was assayed with mice by intraperitoneal injection. LD,o was found to be 8ltg per g . The molecular weight was 13,000 as estimated by gel filtration . The amino acid composition was found to be, Asx1B.,, Thr, . Sers .,, Glx, . Pro, . G1y e .,, Alal .s, 1/2Cysls .2, Val,.,, Met,. 0, lle,.,, Leu, . 9 , Tyr e .,, Phe, . o , Tip,.,, Lyss .1, His, . o , Argp s . Partial amino acid sequence was determined as, Asn .Leu .Try.Gln .Phe.Lys.Asn .Met .lle.Gln . . . . . . . . . MAEDA, N., TAMIYA, N.,' CHEN, Y. M. and LEE, C. Y.,' (1) Department of Chemistry, Tohoku University, Sendai, Japan and (2) Pharmacological Institute, College of Medicine, National Taiwan University, Tapei, Taiwan . THE ISOLATION, PROPERTIESÀND AMINO ACID SEQUENCE OF LATICAUDA SEMIFASCIATA III, A WEAK AND REVERSIBLE NEUROTOXIN FROM THE SEA SNAKE, LATICAUDA SEMI-
FA SCIATA
A new neurotoxin component (Lslll) was isolated by CM-cellulose column chromatography from the venom of the sea snake Laticauda semifasciata. The content of LsIll was about 10-220 per cent of the venom as determined by u .v. absorption at 280 nm . 1sIII was homogeneous on rechromatography and disc electrophoresis, and its molecular weight was 7100 by ultracentrifugation and 7300 by sodium dodecyl sulphate polyacrylamide gel electrophoresis . The fsoelectric point of LsI11 was pH 7-2. LsIll consisted of 66 amino acid residues, including 10 half-cystine residues. The LD SO of LsIll by intramuscular injection was 1-24 ltg per g body weight for mice and 0-45 pg per g for baby chicks, which is about 8-10 times less toxic than erabutoxins a, b and c, all of which are contained in the same venom. Experiments with three isolated muscle preparations from different species indicated that LsIII was a postsynaptically acting toxin, the action of which was easily reversed by washing . The amino acid sequence of LsIII was determined by manual and automatic Edman degradation of the TOXICON 1975 Vol. 13
107
Lim, B.L.1 and SAwAi, Y.,' (1) Division of Medical Ecology, Institute for Medical Research, Kuala Lumpur, Malaysia and (2) The Japan Snake Institute, Yabuzuka Honmachi, Nittagun, Gunma, Japan. ECOLOGY AND DISTRIBUTION OF SEA SNAKES IN PENINSULAR MALAYSIA The sea snakes (Hydrophiinae) have a number of distinctive morphological features which include the reduction of complete degeneration of the ventral shields and a laterally flattened tail like the blade of an oar. All of them are venomous, having poison fangs situated anteriorly in the upper jaw. In Peninsular Malaysia,more than 20 species belonging to 12 genera are known to inhabit the Malaysian waters . TwEmm (1963) grouped these in two subfamilies, the Laticaudinae, with Laticauda colubrina and Aepyurus eydouxi, and Hydrophiinae, comprising all other species. With the exception of Hydrophis, all genera of sea snakes in local waters are represented by a single species. Sea snakes in Peninsular Malaysia are an occasional hazard to fishermen and bathers. A number of fatal cases of sea snake bite have been reported from Penang and other coastal towns (REm et al., 1963 ; Lnw and BAKAR,1970 ; SAwAi et al., 1971). Today very little is known of the ecology and distribution of this group of snakes . The snakes belonging to sub-family Laticaudinae are found in rocky islands and near the shore, hiding in crevices or under stones. Of the two species, Laticauda colubrina is the commonest, this is an amphibious snake, spending part of its time on drier places, particularly in crevices of rocks. Laticauda is the only genus of sea-snakes which is oviparous. Members of the Hydrophiinae can be divided into three different ecological stratifications. There are at least four species of sea snakes known, from the present study, to frequent river mouths, rocky shores, and mangrove shores, and five species are found to inhabit very deep waters usually miles away from the shores. The rest of the species have been known to inhabit deeper waters, but intermediate between the former two groups . It is often difficult for the victim to identify the sea snakes involved with the bite. The treatment of any snakebite depends greatly on the identification of the snake. Knowledge gained from the present study will give an indication of which species of the sea snakes arelikely to be involved depending on the type of areas that the victim visited at the time he was bitten, thus reflecting on the treatment of such cases. Lim, B. L. and DATo Asu BAKAR BIN InAmm (1970). Bites and stings by venomous animals with special reference to snake-bites in West Malaysia. Med. J. Malaya 25,1211-141 . Rte, H. A., TkmAN, P. C. and MARrna, W. T. (1963) Epidemiology of snake-bite in North Malaya . Br . med. J. 7, 992 . SAwAi, Y. and KOBA, K. (1971) Thevenomous snakes of Malaysia. 77re Snake 3,129-152. TwEEDrE, M. W. F. (1961) The Snakes of Malaya. Singapore: Government Printing Office .
LiN, J. Y., Institute of Biochemistry, College of Medicine, National Taiwan University, Tapei, Taiwan . STUDIES ON A NEW CARDIOTOXIN ISOLATED FROM THE EDIBLE MUSHROOM, VOLVARIELLA VOLVACEAE AND FLAMMULINA VELUTIPES Two new cardiotoxic proteins, volvatoxin andfiammutoxin have been isolated from Volvariella volvaceae and F7arnnwlina velutipes, respectively . The molecular weight of fiammutoxin was determined to be 11,000 while volvatoxin consisted of two non-identical subunits volvatoxin A1 and A s, with molecular weight of 23,000 and 50,000, respectively. The LDsu of flamrnutoxin was 2-5 mg per kg body weight and that of volvatoxin was 1-23 mg when the toxic proteins were administered by i.p . igjection into mice. In vitro, both toxic proteins caused the lyses of human erythrocyte in the absence of lecithin, and in vivo they induced EKG changes.
LiN, J. Y., Institute of Biochemistry, College of Medicine, National Taiwan University, Taipei, Taiwan . STUDIES ON ABRIN Abrin was subjected to aminoethylation with ethyleneimine and AE-abrin was fractionated with a CM52 Column Chromatography. Two subunits were obtained, and the molecular weight of the A and B subunit was determined to be 25,000 and 30,000, respectively . Both subunits have no biological activity as compared to native abrin. TO%ICON 1975 Vol. 13
108
4th International Symposium on Animal, Plant and Microbial Toxins
Lint, S . S ., HuANG, M ., TsENG, W. C . and LEE, C. Y., Pharmacological Institute, College of Medicine, National Taiwan University, Taipei, Taiwan. COMPARATIVE STUDIES ON THE MUSCULAR CONTRACTURE INDUCED BY COBRA CARDIOTO-NINN AND SCORPION TOXIN Both cobra cardiotoxin (CTX) and scorpion (Androctonus australis) toxin II are basic polypeptides containing four disulfide bonds ; their molecular weights are 6734 and 7249, respectively . Both toxins are potent in inducing contracture in chick biventer cervicis muscle preparation . Unlike CTX, however, toxin 11 can produce spontaneous contraction in this preparation . LTX contracture was completely abolished whereas toxin II contracture was not affected by high Call (10 mM) . The spontaneous contraction induced by toxin 11 was augmented increasing Ca'+ concentration from 2-5 to 5-0 mM . On the other hand, CTX but not toxin II contracture was accelerated by Cal l free medium . High Mg'+ (10 mM) accelerated CTX contracture but inhibited toxin 11 contracture . Lowering the extracellular Na+ from 143 to 83 mM was without effect on contracture induced by either toxin . Both high Mg'+ and low Na+ did not affect significantly the spontaneous contraction induced by toxin II . Both tetrodotoxin (0-3-3 uM) and procaine (1-8 mM) did not inhibit CT7{ contracture at all but inhibited toxin II contracture markedly. Toxin 11 contracture resumed as soon as these drugs were removed . Both D-tubocurarine (10 - ° g per ml) and ß-bungarotoxin (10 - ' g per ml) had no effect on either CTX or toxin II contracture, but they had a slight inhibitory effect on the spontaneous contraction induced by toxin II. GTX increased "Ca" uptake and caused the inward release of "Ca" in the chick biventer cervicis muscle. Studies on the effect of toxin 11 on "Call fluxes are in progress . The present results suggest that CTX induces contracture by releasing Call from the surface membrane of the muscle, while toxin II may have a different mechanism of action from CTX . As to the spontaneous contraction, toxin II may induce it by acting on the presynaptic region and the muscle membrane, since ß-bungarotoxin can only slightly inhibit it . (Scorpion toxin II was kindly supplied by Prof. F. Miranda, Lab, Biochem . Méd ., Fac . M6d., Marseille.) Lo, T.-B . and CFIANo, W.-C., Institute of Biological Chemistry, Academia Sinica and Institute of Biochemical Sciences, National Taiwan University, Taipei, Taiwan . STUDIES ON PHOSPHOLIPASE A FROM FORMOSAN COBRA (NAJA NAJA ATRA) VENOM Homogeneous phospholipase A from Formosan cobra (Naja naja atra) venom was isolated and purified by CM-cellulose, DEAF-cellulose column chromatography and Sephadex G-50 gel filtration . The specific activity was 400 jig per mg as assayed by titration method with synthetic ß, y-dipalrnito-L-a-lecithin as substrate. This enzyme showed high stability on storing and heating. Apparent Km and Vmax were determined at pH 7-5 and were found to be 9-0 x 10 - ' M and 0-5 lteq per min per ml, respectively . Maximal activity was exhibited at pH 7-1 and at pH 5-2 as measured by turbidimetric method with egg yolk suspension as substrate .Thetoxicity of highly purified phospholipase A was assayed with mice by intraperitoneal injection. LD,o was found to be 8ltg per g . The molecular weight was 13,000 as estimated by gel filtration . The amino acid composition was found to be, Asx1B.,, Thr, . Sers .,, Glx, . Pro, . G1y e .,, Alal .s, 1/2Cysls .2, Val,.,, Met,. 0, lle,.,, Leu, . 9 , Tyr e .,, Phe, . o , Tip,.,, Lyss .1, His, . o , Argp s . Partial amino acid sequence was determined as, Asn .Leu .Try.Gln .Phe.Lys.Asn .Met .lle.Gln . . . . . . . . . MAEDA, N., TAMIYA, N.,' CHEN, Y. M. and LEE, C. Y.,' (1) Department of Chemistry, Tohoku University, Sendai, Japan and (2) Pharmacological Institute, College of Medicine, National Taiwan University, Tapei, Taiwan . THE ISOLATION, PROPERTIESÀND AMINO ACID SEQUENCE OF LATICAUDA SEMIFASCIATA III, A WEAK AND REVERSIBLE NEUROTOXIN FROM THE SEA SNAKE, LATICAUDA SEMI-
FA SCIATA
A new neurotoxin component (Lslll) was isolated by CM-cellulose column chromatography from the venom of the sea snake Laticauda semifasciata. The content of LsIll was about 10-220 per cent of the venom as determined by u .v. absorption at 280 nm . 1sIII was homogeneous on rechromatography and disc electrophoresis, and its molecular weight was 7100 by ultracentrifugation and 7300 by sodium dodecyl sulphate polyacrylamide gel electrophoresis . The fsoelectric point of LsI11 was pH 7-2. LsIll consisted of 66 amino acid residues, including 10 half-cystine residues. The LD SO of LsIll by intramuscular injection was 1-24 ltg per g body weight for mice and 0-45 pg per g for baby chicks, which is about 8-10 times less toxic than erabutoxins a, b and c, all of which are contained in the same venom. Experiments with three isolated muscle preparations from different species indicated that LsIII was a postsynaptically acting toxin, the action of which was easily reversed by washing . The amino acid sequence of LsIII was determined by manual and automatic Edman degradation of the TOXICON 1975 Vol. 13