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Isolation of a purified toxin (Martoxin, MTX) from the venom of the scorpion Buthus martensi Karsch with adrenergic and NANC actions
Abstracts
330
Antigenic characterization of toxin 2 from the venom of the scorpion Centruroides noxius HofSmann. G. Gurrola-Briones, A. Mendez-Teapila and L. D. Possani (Department of Molecular Recognition and Structural Biology, Instituto de Biotecnologia, Universidad National Autonoma de Mexico, Avenida Universidad, 200 1, Apartado Postal 5 10-3, Cuernavaca, Mexico 6227 1). Six different monoclonal antibodies (MAb) were produced against Centruroides noxius toxin 2 (Cn2). one of the most potent toxins from the Mexican scorpions. These MAb define four distinct epitopes in toxin Cn2. The hybridomas named BCF-2 and BCF-3 are capable of producing MAb that neutralize in vivo the effect of 7.5 LD~ doses of Cn2, assayed in mice (Zamudio et al., 1992). Samples of native toxin Cn2 were hydrolysed with enzymes (Staphylococcus aureus V8, trypsin and apartic-N-endopeptidase) and the corresponding peptides were separated by HPLC and assayed using an ELISA format, in order to access recognition of the various HPLC-peptides by the MAb, hence identifying the possible antigenic determinants of toxin Cn2. Digestion with protease V8 produced a fragment (dimeric peptide) corresponding to the amino acid sequences from position 3 to 15 and 54 to 66 (including the disulfide bridge Cys 12_Cys65), which was toxic to mice at the doses assayed (2 pg/20 g mouse weight). This same fragment of Cn2 is recognized by BCF-2 and BCF-3, which seems to indicate the participation of the N-terminal and C-terminal segments of toxin 2 in the toxic effect observed in mice. These findings also suggest that this might explain the in vivo neutralizing capacity of both MAb BCF-2 and BCF-3. Zamudio Supported University
et al. (1992) Eur. I. Biochem. 204, 281-292. in part by grants from Howard Hughes Medical Institute No. 75191-527104, DGAPA-National of Mexico No. 205893 and Consejo National de Ciencia y Tecnologia of Mexico.
Isolation and characterization of insect-specific peptide toxins from the venom of the south Indian red scorpion, Buthus tamulus. W. Rajendra,’ H. Moscowitz,’ R. Hermann,z P. V. Choudary? and B. D. Hammock2 (‘Department of Zoology, Division of Molecular Biology, Sri Venkateswara University, Tirupati, 517 502, A.P., India; and *Department of Entomology and Environmental Toxicology, University of California, Davis, CA 956168584, U.S.A.). Recombinant nuclear polyhedrosis viruses (NPVs) expressing insect selective toxins, hormones or enzymes could enhance the insecticidal properties of the NPVs. Insect specific peptide neurotoxins have been isolated from the venom gland of the south Indian red scorpion, &thus tamulus. The purification of these toxins was performed by CM-cellulose ion-exchange chromatography and HPLC. The degree of purity of the toxins was assessed by capillary electrophoresis and isoelectric focusing. CM-fractions of B. tam&s do not share common epitopes with other known insect selective neurotoxins AaIT, Lqh IT2, Lqh aT and p tox, as revealed by immune cross-reactivity analysis with the antibodies of known scorpion toxins. The toxins (BtITl and BtIT2) induced flaccid paralysis characteristic to depressant neurotoxins in Heliothis virescence larvae, but were non-toxic to Sarcophaga,falculata (blow-fly) larvae and mice. The ecological objective of this study is to construct recombinant baculovirus transfer vectors carrying BtITl and BtIT2 genes and to provide the basis for novel strategies in the development of new biopesticides.
Isolation of a purified toxin (Martoxin, MTX) f rom the venom of the scorpion Buthus martensi Karsch with adrenergic and NANC actions. J.-P. Gong,’ R. Manjunatha Kini, M. C. E. Gwee’ and P. Gopalakrishnakone’ (Venom and Toxin Research Group, Departments of ‘Anatomy and 3Phannacology, Faculty of Medicine, and *Bioscience Centre, Faculty of Science, National University of Singapore, Lower Kent Ridge Road, Singapore 0511). A novel toxin was purified to homogeneity from the crude venom of the scorpion Buthus martensi Karsch (MKV) by a combination of gel filtration on a Bio-gel P-30 column, and cation-exchange chromatography on a POROS HS column, followed by reversed-phase chromatography on a R 2/H 4.6/50 column on a BioCad perfusion chromatography workstation. We have named the toxin Martoxin (MTX). The effects of the toxin (MTX) on adrenergic and non-adrenergic non-cholinergic (NANC) responses were investigated using the rat isolated anococcygeus muscle (Acm) mounted in Krebs solution (37°C 5% CO, in 0,). MTX (10 pg/ml) produced marked and sustained contractions of the Acm which were blocked by phentolamine (5 PM). MTX (IOpg/ml) also markedly relaxed the carbachol precontracted Acm; the relaxation was inhibited by NG-nitro+argininemethylester (L-NAME) (50 PM). Thus, MTX is a novel toxin which can mediate adrenergic as well as NANC responses in the rat Acm. Supported
by research
grants
(RP940304,
RP870357
and RP940325)
from NUS.
330
Antigenic characterization of toxin 2 from the venom of the scorpion Centruroides noxius HofSmann. G. Gurrola-Briones, A. Mendez-Teapila and L. D. Possani (Department of Molecular Recognition and Structural Biology, Instituto de Biotecnologia, Universidad National Autonoma de Mexico, Avenida Universidad, 200 1, Apartado Postal 5 10-3, Cuernavaca, Mexico 6227 1). Six different monoclonal antibodies (MAb) were produced against Centruroides noxius toxin 2 (Cn2). one of the most potent toxins from the Mexican scorpions. These MAb define four distinct epitopes in toxin Cn2. The hybridomas named BCF-2 and BCF-3 are capable of producing MAb that neutralize in vivo the effect of 7.5 LD~ doses of Cn2, assayed in mice (Zamudio et al., 1992). Samples of native toxin Cn2 were hydrolysed with enzymes (Staphylococcus aureus V8, trypsin and apartic-N-endopeptidase) and the corresponding peptides were separated by HPLC and assayed using an ELISA format, in order to access recognition of the various HPLC-peptides by the MAb, hence identifying the possible antigenic determinants of toxin Cn2. Digestion with protease V8 produced a fragment (dimeric peptide) corresponding to the amino acid sequences from position 3 to 15 and 54 to 66 (including the disulfide bridge Cys 12_Cys65), which was toxic to mice at the doses assayed (2 pg/20 g mouse weight). This same fragment of Cn2 is recognized by BCF-2 and BCF-3, which seems to indicate the participation of the N-terminal and C-terminal segments of toxin 2 in the toxic effect observed in mice. These findings also suggest that this might explain the in vivo neutralizing capacity of both MAb BCF-2 and BCF-3. Zamudio Supported University
et al. (1992) Eur. I. Biochem. 204, 281-292. in part by grants from Howard Hughes Medical Institute No. 75191-527104, DGAPA-National of Mexico No. 205893 and Consejo National de Ciencia y Tecnologia of Mexico.
Isolation and characterization of insect-specific peptide toxins from the venom of the south Indian red scorpion, Buthus tamulus. W. Rajendra,’ H. Moscowitz,’ R. Hermann,z P. V. Choudary? and B. D. Hammock2 (‘Department of Zoology, Division of Molecular Biology, Sri Venkateswara University, Tirupati, 517 502, A.P., India; and *Department of Entomology and Environmental Toxicology, University of California, Davis, CA 956168584, U.S.A.). Recombinant nuclear polyhedrosis viruses (NPVs) expressing insect selective toxins, hormones or enzymes could enhance the insecticidal properties of the NPVs. Insect specific peptide neurotoxins have been isolated from the venom gland of the south Indian red scorpion, &thus tamulus. The purification of these toxins was performed by CM-cellulose ion-exchange chromatography and HPLC. The degree of purity of the toxins was assessed by capillary electrophoresis and isoelectric focusing. CM-fractions of B. tam&s do not share common epitopes with other known insect selective neurotoxins AaIT, Lqh IT2, Lqh aT and p tox, as revealed by immune cross-reactivity analysis with the antibodies of known scorpion toxins. The toxins (BtITl and BtIT2) induced flaccid paralysis characteristic to depressant neurotoxins in Heliothis virescence larvae, but were non-toxic to Sarcophaga,falculata (blow-fly) larvae and mice. The ecological objective of this study is to construct recombinant baculovirus transfer vectors carrying BtITl and BtIT2 genes and to provide the basis for novel strategies in the development of new biopesticides.
Isolation of a purified toxin (Martoxin, MTX) f rom the venom of the scorpion Buthus martensi Karsch with adrenergic and NANC actions. J.-P. Gong,’ R. Manjunatha Kini, M. C. E. Gwee’ and P. Gopalakrishnakone’ (Venom and Toxin Research Group, Departments of ‘Anatomy and 3Phannacology, Faculty of Medicine, and *Bioscience Centre, Faculty of Science, National University of Singapore, Lower Kent Ridge Road, Singapore 0511). A novel toxin was purified to homogeneity from the crude venom of the scorpion Buthus martensi Karsch (MKV) by a combination of gel filtration on a Bio-gel P-30 column, and cation-exchange chromatography on a POROS HS column, followed by reversed-phase chromatography on a R 2/H 4.6/50 column on a BioCad perfusion chromatography workstation. We have named the toxin Martoxin (MTX). The effects of the toxin (MTX) on adrenergic and non-adrenergic non-cholinergic (NANC) responses were investigated using the rat isolated anococcygeus muscle (Acm) mounted in Krebs solution (37°C 5% CO, in 0,). MTX (10 pg/ml) produced marked and sustained contractions of the Acm which were blocked by phentolamine (5 PM). MTX (IOpg/ml) also markedly relaxed the carbachol precontracted Acm; the relaxation was inhibited by NG-nitro+argininemethylester (L-NAME) (50 PM). Thus, MTX is a novel toxin which can mediate adrenergic as well as NANC responses in the rat Acm. Supported
by research
grants
(RP940304,
RP870357
and RP940325)
from NUS.