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Purification and characterization of a novel phospholipase A2 from the venom of Conus magus
Fourth International Symposium
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the enzymatic activity of the toxin. Hence, Tyr-21 may be an important residue for the binding of crotoxin and perhaps other phospholipase A 2 neurotoxins. Even stronger evidence comes from the finding that replacement of the corresponding Pbe residue of bovine pancreatic phospholipase A 2 with Tyr by site-directed mutagenesis enables it to compete with an IC50of I #M for the binding of [~25I]crotoxin, contrasting sharply with the complete lack of such ability with the wild-type even at 50/~M. In addition, rat phospholipase A2, which has a corresponding Tyr residue, can inhibit the binding of [125I]crotoxin. TZENG, M.-C. (1993) Interaction of presynaptically toxic phospholipases A 2 with membrane receptors and other binding sites--A review. J. Toxicol.--Toxin Rev. 12, 1~2. Purification and characterization of a novel phospholipase A 2from the venom of Conus magus. S. R. NAISBITT,! B. i . OLIVERA,l S. J. STOEI-IR,2 n . J. DOOLEY,2 F. GHOMASFICHI,3 M. H. GELB3 and J. M. MCINTOSHt'4 (~Dept of Biology, University of Utah, Salt Lake City, UT 84112, U.S.A.; 2Depts of Neuroscience and Chemistry, Parke-Davis, Pharmaceutical Research Div., Warner Lambert, Ann Arbor, MI 48106, U.S.A.; 3Depts of Chemistry and Biochemistry, University of Washington BG-10, Seattle, WA 98195, U.S.A.; and 4Dept of Psychiatry, Univ. of Utah Medical School, Salt Lake City, UT 84132, U.S.A.). PHOSPHOLIPASEA 2 (PLA2) has been found in the venoms of diverse organisms; we present for the first time the purification and characterization of a PLA 2 from the venom of a cone snail. Using several steps of reverse phase HPLC the polypeptide exhibiting PLA 2 activity was isolated from an aqueous extract of Conus magus venom. Molecular weight was determined by electrospray mass spectrophotometry to be approximately 13,700. Reduction yielded two sharp peaks, suggesting disulfide bridged chains. Partial sequence of both chains has been obtained; one contains ~77 residues, the other ~42. Surprisingly, neither chain has appreciable homology to previously sequenced PLA2s. Conus magus PLA 2 (Cm-PLA2) was originally identified because of its effects on ion channels; crude venoms from several species of cone snails were screened independently for ability to inhibit (1) K + depolarization-induced 4SCa uptake into synaptosomes, and (2) binding of [3H]isradipine to L-type Ca 2+ channels. Venom from Conus magus showed potent block in both assays. Using these two assays as markers, the single polypeptide Cm-PLA2 was isolated. PLA 2 is known to release arachidonic acid (AA) and to initiate the AA cascade, which has a regulatory effect on many systems, including L-type Ca 2+ channels. Further studies are being carried out in an effort to determine if specific AA metabolites mediate Cm-PLA2's effects on Ca 2+ channels. Conus magus may not be unique among cone snails in possessing PLA 2 in its venom; we have isolated a polypeptide with similar activity from Conus caracteristicus. This work was supported by grants POI GM48677 (to BMO), HL36235 (to MHG) and K20 MH00929 (to JMM). Ammodytoxin acceptor in bovine brain synaptic membranes: a new approach in the study of the toxic site in ammodytoxins. I. IOUZAJ and F. GUBEN~EK (Department of Biochemistry and Molecular Biology, J. Stefan Institute, Ljubljana, Slovenia). AMMODYTOXINS(ATX) are presynaptically neurotoxic phospholipases A 2 isolated from the venom of long-nosed viper (Vipers ammodytes ammodytes). Isotoxins ATXA and ATXC differ structurally only in two amino acid residues (KRI~AJet al., 1989) but have considerably different toxicities (ATXC is 17 times less toxic than ATXA). They were radioiodinated and used as a ligands. On bovine brain synaptic membranes specific, high affinity, binding sites were demonstrated, t25I-ATXA showed two binding plateaus. Low affinity binding site was not well characterized, while for high affinity binding site Scatchard analysis gave Kd: 3.57 nM and Bmax: 5 pmol/mg of membrane protein. Results obtained for less toxic 12SI-ATXC were Kd: 5.54 nM and Bmax:6 pmol/mg of membrane protein, which correlates with their toxicities. Both toxins were covalently cross-linked to their acceptors in synaptic membranes using bifunctional reagent DMS. Subsequent SDS-PAGE analysis in the presence of DTT followed by autoradiography, revealed a major specific adduct of apparent mol. wt around 70,000. Specific binding was lost when Ca 2+ ions were replaced with SIa+ ions in the incubation buffer. Binding of both toxins, ATXA and ATXC, to the acceptor was strongly inhibited by fl-neurotoxins: crotoxin, crotoxin B and ammodytoxins. Vipers berus betas PLA 2 inhibited binding considerably although it was not reported to be neurotoxic. Ammodytin L, PLA 2 analog with myotoxic action, and ammodytin I2, a non-toxic PLA2, both from Vipers ammodytes ammodytes venom, inhibited binding only at very high concentrations while ~t-dendrotoxin, fl-bungarotoxin and crotoxin A had no influence on binding. The treatment of synaptic membranes with proteinase K and Staphylococcus aureus proteinase lowered the 125I-ATXAand 12~I-ATXC binding, while trypsin treatment had no influence. Heat, acid treatment and incubation of synaptic membranes with mixture of neuraminidase and PNGase F also lowered the binding of t25I-ATXA and ~25I-ATXC. In order to study the 'toxic site' in ammodytoxin, part of the molecule responsible for neurotoxicity (KRIJ~AJet aL, 1989; t~URIN-~ERBECet al., 199 I), the binding characteristics of two ATXA mutants were determined. ATXA (K 98, 101 N) behaved similarly to the native ATXA, while ATXA (K 117 E) binding constants were the same as in the case of ATXC. This is
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the enzymatic activity of the toxin. Hence, Tyr-21 may be an important residue for the binding of crotoxin and perhaps other phospholipase A 2 neurotoxins. Even stronger evidence comes from the finding that replacement of the corresponding Pbe residue of bovine pancreatic phospholipase A 2 with Tyr by site-directed mutagenesis enables it to compete with an IC50of I #M for the binding of [~25I]crotoxin, contrasting sharply with the complete lack of such ability with the wild-type even at 50/~M. In addition, rat phospholipase A2, which has a corresponding Tyr residue, can inhibit the binding of [125I]crotoxin. TZENG, M.-C. (1993) Interaction of presynaptically toxic phospholipases A 2 with membrane receptors and other binding sites--A review. J. Toxicol.--Toxin Rev. 12, 1~2. Purification and characterization of a novel phospholipase A 2from the venom of Conus magus. S. R. NAISBITT,! B. i . OLIVERA,l S. J. STOEI-IR,2 n . J. DOOLEY,2 F. GHOMASFICHI,3 M. H. GELB3 and J. M. MCINTOSHt'4 (~Dept of Biology, University of Utah, Salt Lake City, UT 84112, U.S.A.; 2Depts of Neuroscience and Chemistry, Parke-Davis, Pharmaceutical Research Div., Warner Lambert, Ann Arbor, MI 48106, U.S.A.; 3Depts of Chemistry and Biochemistry, University of Washington BG-10, Seattle, WA 98195, U.S.A.; and 4Dept of Psychiatry, Univ. of Utah Medical School, Salt Lake City, UT 84132, U.S.A.). PHOSPHOLIPASEA 2 (PLA2) has been found in the venoms of diverse organisms; we present for the first time the purification and characterization of a PLA 2 from the venom of a cone snail. Using several steps of reverse phase HPLC the polypeptide exhibiting PLA 2 activity was isolated from an aqueous extract of Conus magus venom. Molecular weight was determined by electrospray mass spectrophotometry to be approximately 13,700. Reduction yielded two sharp peaks, suggesting disulfide bridged chains. Partial sequence of both chains has been obtained; one contains ~77 residues, the other ~42. Surprisingly, neither chain has appreciable homology to previously sequenced PLA2s. Conus magus PLA 2 (Cm-PLA2) was originally identified because of its effects on ion channels; crude venoms from several species of cone snails were screened independently for ability to inhibit (1) K + depolarization-induced 4SCa uptake into synaptosomes, and (2) binding of [3H]isradipine to L-type Ca 2+ channels. Venom from Conus magus showed potent block in both assays. Using these two assays as markers, the single polypeptide Cm-PLA2 was isolated. PLA 2 is known to release arachidonic acid (AA) and to initiate the AA cascade, which has a regulatory effect on many systems, including L-type Ca 2+ channels. Further studies are being carried out in an effort to determine if specific AA metabolites mediate Cm-PLA2's effects on Ca 2+ channels. Conus magus may not be unique among cone snails in possessing PLA 2 in its venom; we have isolated a polypeptide with similar activity from Conus caracteristicus. This work was supported by grants POI GM48677 (to BMO), HL36235 (to MHG) and K20 MH00929 (to JMM). Ammodytoxin acceptor in bovine brain synaptic membranes: a new approach in the study of the toxic site in ammodytoxins. I. IOUZAJ and F. GUBEN~EK (Department of Biochemistry and Molecular Biology, J. Stefan Institute, Ljubljana, Slovenia). AMMODYTOXINS(ATX) are presynaptically neurotoxic phospholipases A 2 isolated from the venom of long-nosed viper (Vipers ammodytes ammodytes). Isotoxins ATXA and ATXC differ structurally only in two amino acid residues (KRI~AJet al., 1989) but have considerably different toxicities (ATXC is 17 times less toxic than ATXA). They were radioiodinated and used as a ligands. On bovine brain synaptic membranes specific, high affinity, binding sites were demonstrated, t25I-ATXA showed two binding plateaus. Low affinity binding site was not well characterized, while for high affinity binding site Scatchard analysis gave Kd: 3.57 nM and Bmax: 5 pmol/mg of membrane protein. Results obtained for less toxic 12SI-ATXC were Kd: 5.54 nM and Bmax:6 pmol/mg of membrane protein, which correlates with their toxicities. Both toxins were covalently cross-linked to their acceptors in synaptic membranes using bifunctional reagent DMS. Subsequent SDS-PAGE analysis in the presence of DTT followed by autoradiography, revealed a major specific adduct of apparent mol. wt around 70,000. Specific binding was lost when Ca 2+ ions were replaced with SIa+ ions in the incubation buffer. Binding of both toxins, ATXA and ATXC, to the acceptor was strongly inhibited by fl-neurotoxins: crotoxin, crotoxin B and ammodytoxins. Vipers berus betas PLA 2 inhibited binding considerably although it was not reported to be neurotoxic. Ammodytin L, PLA 2 analog with myotoxic action, and ammodytin I2, a non-toxic PLA2, both from Vipers ammodytes ammodytes venom, inhibited binding only at very high concentrations while ~t-dendrotoxin, fl-bungarotoxin and crotoxin A had no influence on binding. The treatment of synaptic membranes with proteinase K and Staphylococcus aureus proteinase lowered the 125I-ATXAand 12~I-ATXC binding, while trypsin treatment had no influence. Heat, acid treatment and incubation of synaptic membranes with mixture of neuraminidase and PNGase F also lowered the binding of t25I-ATXA and ~25I-ATXC. In order to study the 'toxic site' in ammodytoxin, part of the molecule responsible for neurotoxicity (KRIJ~AJet aL, 1989; t~URIN-~ERBECet al., 199 I), the binding characteristics of two ATXA mutants were determined. ATXA (K 98, 101 N) behaved similarly to the native ATXA, while ATXA (K 117 E) binding constants were the same as in the case of ATXC. This is