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Fluorescent labeled α-conotoxin GI: binding interactions with the nicotinic acetylcholine receptor and monoclonal antibodies
Abstracts
301
Fluorescent labeled cc-conotoxin GI: binding interactions with the nicotinic acetylcholine receptor and monoclonal antibodies. J. D. Ashcom and B. G. Stiles (Fort Detrick USAMRIID Toxinology Division, Frederick, MD 21702,
U.S.A.). The a-conotoxins, obtained in nature from the venom of predatory marine snails, are a structurally related family of low mol. wt polypeptides which bind to the nicotinic acetylcholine receptor and cause rapid muscular paralysis and death in animals. These peptides contain 13-15 amino acids with two intramolecular disulfide bonds. A fluorescent-labeled derivative of a-conotoxin GI has been prepared which binds with the receptor and also with protective monoclonal antibodies. Unlabeled ligands block the specific binding of the fluorescent peptide. This material has been used in a quantitative binding assay system which features solution phase interaction with conotoxin binding proteins followed by the rapid separation of unbound ligand. The fluorescent signal is linearly dependent on ligand concentration over a wide range, and calibration curves permit the determination of molar binding ratios. These techniques could be used to measure low mol. wt ligand binding interactions in other systems. Novel primary
struclure
of sticholysin
V. Besada,z F. Pazos,’ M. E. Lanio’ Gen&tica y Biotechnologia, Havana,
and its interaction
and G. Pad&? Cuba).
with membranes.
(‘Universidad
C. Alvarez,’ M. Tejuca,’ V. Morera, de La Habana and ‘Centro de Ingenieria
Sticholysin-I (St-I) is a basic 18,000 mol. wt polypeptide purified from the anemone Stichodactyla hefianthus that exhibits haemolytic activity (HC,, 25-30 ng/ml). Amino acid sequencing showed the same amino acids up to cycle 29 as C-III (Blumenthal and Kern, 1983). However, from cycle 30 to 51 the amino acids detected did not match with those of C-III, but they were identical again after cycle 51 to 67. Sequence alignments revealed more than 65% homology with Eq-II from Actinia equina, which also contains the 22 amino acid insert. St-I forms a hydrophilic pore of 1 nm internal radius. Induced haemoglobin or calcein release from cells or LUV-liposomes exhibited a Hill coefficient > I, indicating cooperativity among St-I monomers. Nevertheless, preincubation of St-I in a saline medium provoked a decrease in cooperativity. Evaluation of conformational changes could not detect any difference between the polypeptide preincubated or not in saline, suggesting that potentiation is not mediated by preaggregation in solution. The enhancing role of the medium ionic strength on the haemolytic capacity of St-I might be related with a transition to a more relaxed conformation that could interact more efficiently with membranes demanding less confluence of other St-1 monomers to cause lysis. PuriJication, characterizarion and immobilization oj‘proteinase inhibitors from Stichodactyla helianthus. J. Delfin,’ and J. Diaz,’ W. Antuch,2 R. Rodriguez,’ Y. GonzBlez, ’ V. Morera,* I. Martinez,’ N. Larionova,3 G. Pad&’ M. ChBvez’ (‘Facultad de Biologia, Universidad de La Habana, Cuba; 2Centro de Ingenieria Genetica y Biotecnologia, Habana, Cuba; and 3Faculty of Chemistry, Moscow State University, Russia).
The existence of proteinase inhibitors from Sroichactis sp. (Sfichodactyla sp.) has been reported (Mebs and Gebauer, 1980; Chavez et a/., 1985). Proteinase inhibitors were isolated from the sea anemone Stichodactyla helianthus. The aqueous extract was previously treated with trichloroacetic acid followed by affinity chromatography on Trypsin-Sepharose and either gel filtration on Sephadex G-50 or ion-exchange chromatography on CM-Cellulose. The isoelectric point of the major inhibitor (ShPI) is 8.4 and the average mol. wt obtained by fast atom bombardment (FAB-MS) is 6110. The amino acid sequence was determined by FAB-MS combined with manual Edman degradation, endo and exo peptidases treatments and automatic sequencing. The sequence of ShPI (55 amino acids) was compared with those reported in the Protein Data Bank for several proteinase inhibitors; significant similarity to inhibitors belonging to the Kunitz family was observed. ShPI exhibits a broad specificity for serine, cysteine and aspartic proteinases. The dissociation constant of the complexes formed with different enzymes were determined. The affinity purified fraction was immobilized in different supports and similar results were obtained with Sepharose and Cellulose. Insecf
venom allergens.
T. P. King (Rockefeller
University,
New York,
NY 10021, U.S.A.).
Insect sting allergies to fire ants, bees and vespids are common in U.S. The vespids include hornets, yellowjackets and wasps. Susceptible people can become sensitized following a single sting of < 10 pg of venom proteins and they develop venom-specific IgEs. The major allergens from these insects have been sequenced and/or cloned by various investigators. Our studies are mainly with the vespid allergens. Insect allergens are proteins of 120-340 amino acid residues. Some insect allergens are structurally and biochemically similar but some are not. For example, both bee and vespid venoms contain hyaluronidases with a high degree of sequence similarity but their phospholipases differ markedly in their sequences and their enzymatic specificities. Another venom protein of unknown biological function, designated as antigen 5, is present in vespids and fireants but not in bees. One common feature of the venom allergens is their varying extents of sequence similarity with other proteins in our
301
Fluorescent labeled cc-conotoxin GI: binding interactions with the nicotinic acetylcholine receptor and monoclonal antibodies. J. D. Ashcom and B. G. Stiles (Fort Detrick USAMRIID Toxinology Division, Frederick, MD 21702,
U.S.A.). The a-conotoxins, obtained in nature from the venom of predatory marine snails, are a structurally related family of low mol. wt polypeptides which bind to the nicotinic acetylcholine receptor and cause rapid muscular paralysis and death in animals. These peptides contain 13-15 amino acids with two intramolecular disulfide bonds. A fluorescent-labeled derivative of a-conotoxin GI has been prepared which binds with the receptor and also with protective monoclonal antibodies. Unlabeled ligands block the specific binding of the fluorescent peptide. This material has been used in a quantitative binding assay system which features solution phase interaction with conotoxin binding proteins followed by the rapid separation of unbound ligand. The fluorescent signal is linearly dependent on ligand concentration over a wide range, and calibration curves permit the determination of molar binding ratios. These techniques could be used to measure low mol. wt ligand binding interactions in other systems. Novel primary
struclure
of sticholysin
V. Besada,z F. Pazos,’ M. E. Lanio’ Gen&tica y Biotechnologia, Havana,
and its interaction
and G. Pad&? Cuba).
with membranes.
(‘Universidad
C. Alvarez,’ M. Tejuca,’ V. Morera, de La Habana and ‘Centro de Ingenieria
Sticholysin-I (St-I) is a basic 18,000 mol. wt polypeptide purified from the anemone Stichodactyla hefianthus that exhibits haemolytic activity (HC,, 25-30 ng/ml). Amino acid sequencing showed the same amino acids up to cycle 29 as C-III (Blumenthal and Kern, 1983). However, from cycle 30 to 51 the amino acids detected did not match with those of C-III, but they were identical again after cycle 51 to 67. Sequence alignments revealed more than 65% homology with Eq-II from Actinia equina, which also contains the 22 amino acid insert. St-I forms a hydrophilic pore of 1 nm internal radius. Induced haemoglobin or calcein release from cells or LUV-liposomes exhibited a Hill coefficient > I, indicating cooperativity among St-I monomers. Nevertheless, preincubation of St-I in a saline medium provoked a decrease in cooperativity. Evaluation of conformational changes could not detect any difference between the polypeptide preincubated or not in saline, suggesting that potentiation is not mediated by preaggregation in solution. The enhancing role of the medium ionic strength on the haemolytic capacity of St-I might be related with a transition to a more relaxed conformation that could interact more efficiently with membranes demanding less confluence of other St-1 monomers to cause lysis. PuriJication, characterizarion and immobilization oj‘proteinase inhibitors from Stichodactyla helianthus. J. Delfin,’ and J. Diaz,’ W. Antuch,2 R. Rodriguez,’ Y. GonzBlez, ’ V. Morera,* I. Martinez,’ N. Larionova,3 G. Pad&’ M. ChBvez’ (‘Facultad de Biologia, Universidad de La Habana, Cuba; 2Centro de Ingenieria Genetica y Biotecnologia, Habana, Cuba; and 3Faculty of Chemistry, Moscow State University, Russia).
The existence of proteinase inhibitors from Sroichactis sp. (Sfichodactyla sp.) has been reported (Mebs and Gebauer, 1980; Chavez et a/., 1985). Proteinase inhibitors were isolated from the sea anemone Stichodactyla helianthus. The aqueous extract was previously treated with trichloroacetic acid followed by affinity chromatography on Trypsin-Sepharose and either gel filtration on Sephadex G-50 or ion-exchange chromatography on CM-Cellulose. The isoelectric point of the major inhibitor (ShPI) is 8.4 and the average mol. wt obtained by fast atom bombardment (FAB-MS) is 6110. The amino acid sequence was determined by FAB-MS combined with manual Edman degradation, endo and exo peptidases treatments and automatic sequencing. The sequence of ShPI (55 amino acids) was compared with those reported in the Protein Data Bank for several proteinase inhibitors; significant similarity to inhibitors belonging to the Kunitz family was observed. ShPI exhibits a broad specificity for serine, cysteine and aspartic proteinases. The dissociation constant of the complexes formed with different enzymes were determined. The affinity purified fraction was immobilized in different supports and similar results were obtained with Sepharose and Cellulose. Insecf
venom allergens.
T. P. King (Rockefeller
University,
New York,
NY 10021, U.S.A.).
Insect sting allergies to fire ants, bees and vespids are common in U.S. The vespids include hornets, yellowjackets and wasps. Susceptible people can become sensitized following a single sting of < 10 pg of venom proteins and they develop venom-specific IgEs. The major allergens from these insects have been sequenced and/or cloned by various investigators. Our studies are mainly with the vespid allergens. Insect allergens are proteins of 120-340 amino acid residues. Some insect allergens are structurally and biochemically similar but some are not. For example, both bee and vespid venoms contain hyaluronidases with a high degree of sequence similarity but their phospholipases differ markedly in their sequences and their enzymatic specificities. Another venom protein of unknown biological function, designated as antigen 5, is present in vespids and fireants but not in bees. One common feature of the venom allergens is their varying extents of sequence similarity with other proteins in our