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New polar water-soluble cytotoxins from echinoderms
Tenth World Congress
505
Purification and partial characterization o f viperabin a thrombin-like enzmye from the venom o f Cerastes vipera (Sahara sand viper). M. F. EL-ASMAR, T. M. FARID and H. NASSER (Department of Biochemistry, Faculty of Medicine, Ain Shams University, Cairo, Egypt). VIPERABIN, a thrombin-like enzyme, was isolated from the crude venom of Cerastes vipera in homogenous form,
by three successive steps of chromatographic techniques. It had a mol. wt of 32,000, and an isoelectric point of 4.9. It had no carbohydrate content, and showed no arginine esterase activity nor proteolytic activity on albumin or casein. It produced fibrin from fibrinogen and hydrolysed the chromogenic substrate for thrombin (CBS 34.47). Viperabin clotted fibrinogen with a specific activity of 12.5 NIH thrombin equivalent units/mg. Viperabin potentiated the coagulation of normal plasma as well as plasmas deficient in factors II, V, VII, VIII, IX, X, XI, XII, or XIII. The thrombin-like activity of viperabin was markedly inhibited by leupeptin, DEP and iodoacetamide. This may indicate a serine protease with an active thiol group. Viperabin had a moderate platelet aggregatory effect. Further characterization o f cerastobin from Cerastes vipera (Egyptian Sahara sand viper) venom. M . F . EL-ASMAR,I T.M. FARIDl and A. Tu 2 (IDepartment of Biochemistry, Faculty of Medicine, Ain Shams University, Cairo, Egypt; 2Colorado State University, Fort Collins, Colorado, U.S.A.). CERASTOBIN purified from Cerastes vipera venom shortened the clotting time of normal citrated plasma in the presence of calcium, and caused coagulation even in the absence of calcium. It also clotted plasmas deficient in factors II, V, VII, VIII, IX, XI or XIII. A fibrin clot was produced by the effect of cerastobin on pure fibrinogen. Heparin blocked the coagulant effect of cerastobin. The course of increase in turbidity of a fibrinogen solution by cerastobin is different from that of thrombin. The maximal rate of assembly of the clot is also different. Cerastobin could partially hydrolyse prothrombin to form possibly prethrombin-2. Cerastobin could attack the Arg-Gly bond in the oxidized B-chain of insulin. It could also attack other bonds in the same substrate, although at slower rates. Purification and characterization of a protease active on rabbit platelets purified from Cerastes cerastes venom. M. EL AYEB,t N. MARRAKCH1,I L. Z1NGHALI,2 C. BON2 and K. DELLAGII (~Laboratoire, Venins et Toxines, Institut Pasteur de Tunis BP. 74-1002, Tunis; 2Service des Venins, Institut Pasteur de Paris, France). A SINGLE-chain glycoprotein with mol. wt 38,000 has been purified on Mono O and Mono S FPLC columns. This protein shows amodolytic activity on the chromogenic substrate of the thrombin ($2238), and is able to aggregate washed rabbit platelets. Moreover, a low procoagulant activity is exhibited. The characterization of this protein with enzyme specific inhibitors revealed that it is a serine protease. The conformation and the thiol groups are important for the expression of the activity. The use of platelet specific inhibitors indicated that at least two pathways of activation are involved. New polar water-soluble cytotoxinsfrom echinoderms. G. B. ELYAKOV,V. A. STONIK and S. N. FEDOROV(Pacific Institute of Bioorganic Chemistry of the U.S.S.R. Academy of Sciences, Vladivostok-22, Russia). AMONG aquatic organisms, echinoderms contain a great variety of polar, water-soluble natural products such as organic sulphates. The majority of these metabolites are steroidal cytotoxins, which are considered to play a role in chemical defence against predators and different pathogenic swimming microforms. At present compounds from ophiuroids have contributed little to the increasing number of polar molecules from holothurians and starfish. A series of new physiologically active steroid sulphates, identified as (20R)-cholesta-5, 22E-dien-3ct, 4fl, 21-triol 3, 21-disulphate; (20R)-cholesta-5, 25-dien-3~t, 4fl, 21-triol 3, 21-disulphate; (20R, 24R)-cholesta-5, 25dien-2fl, 3~, 21, 24-tetrol 3, 21-disulphate; (20R, 24S)-cholesta-5, 25-dien-2fl, 3~t, 21, 24-tetrol 3, 21-disulphate; and (20R)-24-norcholesta-5, 22E-dien-2fl, 3~t, 21-triol 3, 21-disulphate, has been isolated from the far-eastern ophiuroid Ophiopholis aculeata. All these secondary metabolites are 3, 21-disulphates having the 5(6)-double bond. Cytotoxic action of these compounds depends upon the structures of both the polycyclic system and the side chain moieties. Degradation o f tetanus and botulinum A neurotoxins in chroma~n cells. G. ERDMANN, E. ERDAL, F. BARTEI.Sand H. BIGALKE(Department of Toxicology, Medical School of Hannover, 3000 Hannover, F.R.G.). TETANUS and botulinum A neurotoxins inhibit noradrenaline release from bovine adrenal chromatfin cells in vitro. The blocking effects of the toxins persist for several days. Restoration of exocytosis, however, can be achieved within a few days if tetanus toxin is intracellularly neutralized by its specific antibodies. Physiologically, antibodies cannot pass through the plasma membrane of neurons and chromaffin cells. Nevertheless, tetanus and
506
Tenth World Congress
botulism are reversible in vivo, although the diseases are long-lasting. Since the pharmacodynamics and time courses of both toxins are of striking similarity in neurons and in chromaffin cells we wondered how the cells handle these proteins and by what means recovery of the physiological functions is brought about. For this purpose, chromaffin cells were electroporated in the presence of t25I-tetanus toxin, t25I-botulinum A neurotoxin, or tetanus toxin bound to colloidal gold particles with a diameter of approximately 5 nm. During electroporation these labelled toxins diffused into the cytosol without binding to the cell surface, because chromaffin cells lack toxin receptors. Intracellular localization of gold-labelled toxin could be confirmed by optical methods. Both m-labelled toxins were split intracetlularly into their heavy and light chains. This degradation is a prerequisite for the occurrence of the toxic effects, because only the separated light chains contain the intrinsic activity. The chains were further degraded into smaller fragments, the light chains being more resistant to degradation than the heavy chains. After about 5 days only fragments with a mol. wt below 15,000 were detectable, while exocytosis continued to be blocked. We conclude from these findings that either a fragment smaller than the light chains conserves the active site of toxins or, alternatively, that after the complete disappearance of the light chains exocytosis recovers only after the intracellular targets of the toxins have been resynthesized. (Supported by the DFG.)
Isolation and partial characterization of a fibrinogenase from the venom o f the Peruvian bushmaster snake Lachesis muta. E. ESCOBAR,E. RODRIGUEZ and A. YARLEQtr~ (Laboratory of Molecular Biology, ICBAR, Faculty of Biological Sciences, San Marcos University, Lima, Peru). A FIBRINOGENASE has been isolated from the crude venom of Lachesis mum using DEAE Sephadex A-50 column equilibrated with 0.05 M Tris-HC1 buffer, pH 7.5, and gel filtration on Sephadex G-75 with the same buffer containing 0.15M sodium chloride solution. In both steps the enzyme was recovered with the first peak of protein, measuring its activity by delay of clotting. In contrast to proteinase I and II described previously by us in the same venom, fibrinogenase does not attack casein. Synthetic ester substrates (TAME and BAEE) and synthetic amides (BAPNA) are not sensitive to enzymatic action. A single protein band was observed on PAGE-SDS, pH 7.0, with a mol. wt of 83,800. The mode of action on bovine fibrinogen was investigated with PAGE-SDS, pH 7.0, using fibrinogenase as well as proteinase I and II. Fibrinogen solution (4.5 mg/ml) in 0.05 M Tris-HC1 buffer, pH 7.5, was incubated with aliquots of each enzyme during 5-120 min and 20 #1 was denatured with sample buffer and applied on the PAGE-SDS system. Our results showed that three enzymes attack bovine fibrinogen through Act chains initially and Bfl chains after 30 min of incubation, while chains were not affected. However, differences among these enzymes were observed by hydrolysis products, which were more abundant in the case of proteinasc I than proteinase II, and a few products were found in the case of fibrinogenase. We assume that these three enzymes have an important role in coagulation disorders during envenomation caused by this snake. Additional results on human fibrinogen will be presented.
Invertebrate specific neurotoxins from non-piscivorous Conus venoms. M. FAINZILBER, D. ~ORDON and E. ZLOTrdN (Institute of Life Sciences, Hebrew University of Jerusalem; Interuniversity Institute, Eilat, Israel). THE CONIDAE are a family of venomous marine snails subdivided according to their feeding ecology into piscivorous, molluscivorous and vermivorous species. In this study we report the purification and characterization of invertebrate specific neurotoxins from the venoms of non-piscivorous Conidae from the Red Sea. New bioassays for paralysis in molluscs and annelids were developed and used for screening the venoms of 12 species of Conidae of all three feeding specificities. A clear correlation was found between the feeding specificity of the Conidae studied and the phylogenic specificity of their crude venoms. Piscivorous species venoms (Conus striatus, C. nigropanctatus) possessed high toxicity to vertebrates and ten to 30-fold weaker effects in invertebrates. Venoms from five vermivorous species had no effects in vertebrates or molluscs, but caused localized contractions in annelids. The venoms of two molluscivorous species (C. textile, C. pennaceus) were found to be strictly selective for molluscs. Conus venoms were fractionated by Scphadex G-50 chromatography followed by RP-HPLC. This has resulted in the purification of a number of peptides with specific toxicity for molluscs or annelids. Characterization of three mollusc specific peptide toxins from C. textile has shown that they are typically small (25-27 residues) cysteine-rich conotoxins. They differ from piscivorous venom conotoxins in their net negative charge and higher hydrophobicity. One especially interesting toxin contains an uneven number of cysteines and two gamma-carboxyghitamate residues. This extremely negatively charged toxin is the most potent of the mollusc paralysing conotoxins. The three C. textile conotoxins increase excitability of Aplysia neurons, in contrast to the blocking actions of all previously characterized conotoxins. The mollusc or annelid specific conotoxins should prove to be useful probes for unique features of invertebrate nervous systems.
505
Purification and partial characterization o f viperabin a thrombin-like enzmye from the venom o f Cerastes vipera (Sahara sand viper). M. F. EL-ASMAR, T. M. FARID and H. NASSER (Department of Biochemistry, Faculty of Medicine, Ain Shams University, Cairo, Egypt). VIPERABIN, a thrombin-like enzyme, was isolated from the crude venom of Cerastes vipera in homogenous form,
by three successive steps of chromatographic techniques. It had a mol. wt of 32,000, and an isoelectric point of 4.9. It had no carbohydrate content, and showed no arginine esterase activity nor proteolytic activity on albumin or casein. It produced fibrin from fibrinogen and hydrolysed the chromogenic substrate for thrombin (CBS 34.47). Viperabin clotted fibrinogen with a specific activity of 12.5 NIH thrombin equivalent units/mg. Viperabin potentiated the coagulation of normal plasma as well as plasmas deficient in factors II, V, VII, VIII, IX, X, XI, XII, or XIII. The thrombin-like activity of viperabin was markedly inhibited by leupeptin, DEP and iodoacetamide. This may indicate a serine protease with an active thiol group. Viperabin had a moderate platelet aggregatory effect. Further characterization o f cerastobin from Cerastes vipera (Egyptian Sahara sand viper) venom. M . F . EL-ASMAR,I T.M. FARIDl and A. Tu 2 (IDepartment of Biochemistry, Faculty of Medicine, Ain Shams University, Cairo, Egypt; 2Colorado State University, Fort Collins, Colorado, U.S.A.). CERASTOBIN purified from Cerastes vipera venom shortened the clotting time of normal citrated plasma in the presence of calcium, and caused coagulation even in the absence of calcium. It also clotted plasmas deficient in factors II, V, VII, VIII, IX, XI or XIII. A fibrin clot was produced by the effect of cerastobin on pure fibrinogen. Heparin blocked the coagulant effect of cerastobin. The course of increase in turbidity of a fibrinogen solution by cerastobin is different from that of thrombin. The maximal rate of assembly of the clot is also different. Cerastobin could partially hydrolyse prothrombin to form possibly prethrombin-2. Cerastobin could attack the Arg-Gly bond in the oxidized B-chain of insulin. It could also attack other bonds in the same substrate, although at slower rates. Purification and characterization of a protease active on rabbit platelets purified from Cerastes cerastes venom. M. EL AYEB,t N. MARRAKCH1,I L. Z1NGHALI,2 C. BON2 and K. DELLAGII (~Laboratoire, Venins et Toxines, Institut Pasteur de Tunis BP. 74-1002, Tunis; 2Service des Venins, Institut Pasteur de Paris, France). A SINGLE-chain glycoprotein with mol. wt 38,000 has been purified on Mono O and Mono S FPLC columns. This protein shows amodolytic activity on the chromogenic substrate of the thrombin ($2238), and is able to aggregate washed rabbit platelets. Moreover, a low procoagulant activity is exhibited. The characterization of this protein with enzyme specific inhibitors revealed that it is a serine protease. The conformation and the thiol groups are important for the expression of the activity. The use of platelet specific inhibitors indicated that at least two pathways of activation are involved. New polar water-soluble cytotoxinsfrom echinoderms. G. B. ELYAKOV,V. A. STONIK and S. N. FEDOROV(Pacific Institute of Bioorganic Chemistry of the U.S.S.R. Academy of Sciences, Vladivostok-22, Russia). AMONG aquatic organisms, echinoderms contain a great variety of polar, water-soluble natural products such as organic sulphates. The majority of these metabolites are steroidal cytotoxins, which are considered to play a role in chemical defence against predators and different pathogenic swimming microforms. At present compounds from ophiuroids have contributed little to the increasing number of polar molecules from holothurians and starfish. A series of new physiologically active steroid sulphates, identified as (20R)-cholesta-5, 22E-dien-3ct, 4fl, 21-triol 3, 21-disulphate; (20R)-cholesta-5, 25-dien-3~t, 4fl, 21-triol 3, 21-disulphate; (20R, 24R)-cholesta-5, 25dien-2fl, 3~, 21, 24-tetrol 3, 21-disulphate; (20R, 24S)-cholesta-5, 25-dien-2fl, 3~t, 21, 24-tetrol 3, 21-disulphate; and (20R)-24-norcholesta-5, 22E-dien-2fl, 3~t, 21-triol 3, 21-disulphate, has been isolated from the far-eastern ophiuroid Ophiopholis aculeata. All these secondary metabolites are 3, 21-disulphates having the 5(6)-double bond. Cytotoxic action of these compounds depends upon the structures of both the polycyclic system and the side chain moieties. Degradation o f tetanus and botulinum A neurotoxins in chroma~n cells. G. ERDMANN, E. ERDAL, F. BARTEI.Sand H. BIGALKE(Department of Toxicology, Medical School of Hannover, 3000 Hannover, F.R.G.). TETANUS and botulinum A neurotoxins inhibit noradrenaline release from bovine adrenal chromatfin cells in vitro. The blocking effects of the toxins persist for several days. Restoration of exocytosis, however, can be achieved within a few days if tetanus toxin is intracellularly neutralized by its specific antibodies. Physiologically, antibodies cannot pass through the plasma membrane of neurons and chromaffin cells. Nevertheless, tetanus and
506
Tenth World Congress
botulism are reversible in vivo, although the diseases are long-lasting. Since the pharmacodynamics and time courses of both toxins are of striking similarity in neurons and in chromaffin cells we wondered how the cells handle these proteins and by what means recovery of the physiological functions is brought about. For this purpose, chromaffin cells were electroporated in the presence of t25I-tetanus toxin, t25I-botulinum A neurotoxin, or tetanus toxin bound to colloidal gold particles with a diameter of approximately 5 nm. During electroporation these labelled toxins diffused into the cytosol without binding to the cell surface, because chromaffin cells lack toxin receptors. Intracellular localization of gold-labelled toxin could be confirmed by optical methods. Both m-labelled toxins were split intracetlularly into their heavy and light chains. This degradation is a prerequisite for the occurrence of the toxic effects, because only the separated light chains contain the intrinsic activity. The chains were further degraded into smaller fragments, the light chains being more resistant to degradation than the heavy chains. After about 5 days only fragments with a mol. wt below 15,000 were detectable, while exocytosis continued to be blocked. We conclude from these findings that either a fragment smaller than the light chains conserves the active site of toxins or, alternatively, that after the complete disappearance of the light chains exocytosis recovers only after the intracellular targets of the toxins have been resynthesized. (Supported by the DFG.)
Isolation and partial characterization of a fibrinogenase from the venom o f the Peruvian bushmaster snake Lachesis muta. E. ESCOBAR,E. RODRIGUEZ and A. YARLEQtr~ (Laboratory of Molecular Biology, ICBAR, Faculty of Biological Sciences, San Marcos University, Lima, Peru). A FIBRINOGENASE has been isolated from the crude venom of Lachesis mum using DEAE Sephadex A-50 column equilibrated with 0.05 M Tris-HC1 buffer, pH 7.5, and gel filtration on Sephadex G-75 with the same buffer containing 0.15M sodium chloride solution. In both steps the enzyme was recovered with the first peak of protein, measuring its activity by delay of clotting. In contrast to proteinase I and II described previously by us in the same venom, fibrinogenase does not attack casein. Synthetic ester substrates (TAME and BAEE) and synthetic amides (BAPNA) are not sensitive to enzymatic action. A single protein band was observed on PAGE-SDS, pH 7.0, with a mol. wt of 83,800. The mode of action on bovine fibrinogen was investigated with PAGE-SDS, pH 7.0, using fibrinogenase as well as proteinase I and II. Fibrinogen solution (4.5 mg/ml) in 0.05 M Tris-HC1 buffer, pH 7.5, was incubated with aliquots of each enzyme during 5-120 min and 20 #1 was denatured with sample buffer and applied on the PAGE-SDS system. Our results showed that three enzymes attack bovine fibrinogen through Act chains initially and Bfl chains after 30 min of incubation, while chains were not affected. However, differences among these enzymes were observed by hydrolysis products, which were more abundant in the case of proteinasc I than proteinase II, and a few products were found in the case of fibrinogenase. We assume that these three enzymes have an important role in coagulation disorders during envenomation caused by this snake. Additional results on human fibrinogen will be presented.
Invertebrate specific neurotoxins from non-piscivorous Conus venoms. M. FAINZILBER, D. ~ORDON and E. ZLOTrdN (Institute of Life Sciences, Hebrew University of Jerusalem; Interuniversity Institute, Eilat, Israel). THE CONIDAE are a family of venomous marine snails subdivided according to their feeding ecology into piscivorous, molluscivorous and vermivorous species. In this study we report the purification and characterization of invertebrate specific neurotoxins from the venoms of non-piscivorous Conidae from the Red Sea. New bioassays for paralysis in molluscs and annelids were developed and used for screening the venoms of 12 species of Conidae of all three feeding specificities. A clear correlation was found between the feeding specificity of the Conidae studied and the phylogenic specificity of their crude venoms. Piscivorous species venoms (Conus striatus, C. nigropanctatus) possessed high toxicity to vertebrates and ten to 30-fold weaker effects in invertebrates. Venoms from five vermivorous species had no effects in vertebrates or molluscs, but caused localized contractions in annelids. The venoms of two molluscivorous species (C. textile, C. pennaceus) were found to be strictly selective for molluscs. Conus venoms were fractionated by Scphadex G-50 chromatography followed by RP-HPLC. This has resulted in the purification of a number of peptides with specific toxicity for molluscs or annelids. Characterization of three mollusc specific peptide toxins from C. textile has shown that they are typically small (25-27 residues) cysteine-rich conotoxins. They differ from piscivorous venom conotoxins in their net negative charge and higher hydrophobicity. One especially interesting toxin contains an uneven number of cysteines and two gamma-carboxyghitamate residues. This extremely negatively charged toxin is the most potent of the mollusc paralysing conotoxins. The three C. textile conotoxins increase excitability of Aplysia neurons, in contrast to the blocking actions of all previously characterized conotoxins. The mollusc or annelid specific conotoxins should prove to be useful probes for unique features of invertebrate nervous systems.