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Studies of the presynaptic effect of β-bungarotoxin on neuromuscular transmission
FRLSCHAUF, A: M. and Ecxsran~, F. (Max Planck Institut of Biophysics and Chemistry, Gottingen 3400, West Germany) . Purification of a phosphodiesterase from Bothrops atrox venom by affinity chromatography. J. Biochem. 32, 479, 1973 .
TsE ruxtFlcwnoty of a phosphodiesterase from Bothrops atrox venom by chromatography on phosphocellulose and affinity chromatography with O-(4-nitrophenyl}O'-phenyl-thiophosphate ester coupled to activated Sepharose is described. Phosphatases are removed by chromatography on hydroxyapatite . The enzyme is a single-chain protein with a molecular weight of approximately 130,01x1 as judged by dodecylsulfate-gel electrophoresis and ultracentrifugation. It has a specific activity of 3~3 pm bis (4-nitrophenyl) phosphate hydrolyzed min'' mg 1 at 24°C . W.F.K .
Toots, P. M. and Cxwta, D. S. (Dept. of Chemistry, University of Southern Mississippi, Hattiesburg, Mississippi 39401, U.S .A .). Enzymatic activities of venom from the jellyfish Stomolophus meleagris. Comp . Biochem. Physiol. 43B, 435, 1972 .
venom from the nematocysts of the jellyfish Stomolophus meleagris was tested for enzymatic activity on 28 substrates commonly hydrolyzed by many venoms. Twelve of the substrates tested were hydrolyzed, suggesting the presence of 5'-nucleotidase, hyaluronidase, acid and alkaline phosphatases, phosphodiesterase, leucine aminopeptidase and professes, all of which have also been found in snake venoms . On the other hand, a number of enzymes found in some snake venoms (e.g . acetylcholine esterase, blood clotting enzymes, amino acid oxidases, amino acid esterases, and phospholipases) were not detectod in venom of S. meleagris. The enzymatic nature and physiological actions of jellyfish venom are compared with those of snake venoms . P.R.S. LYOP~-t~~
Ct3.~,taa, C. C. and Y~tvct, C. C. (Dept. of Biochem., Kaohsiung Medical College, Kaohsiung, Taiwan, Republic of China) . Tmmunochemical studies on the tryptophan-modified cobrotoxin . Blochim. biophys. Acta 295, 595, 1973).
Mosr sruxE neurotoxins whose amino acid sequences have been established contain only one Trp-residue located at the homologous position. The single Trp-residue in cobrotoxin, a neurotoxic protein obtained from the venom of Taiwan cobra (NgJa ngja atra), has been converted into N-formylkynurenine by ozonization in formic acid, oxidised to oxindole derivative with N-bromosuocinimide, and also modified by reactions with 2-hydroxy-5-nitrobenzyl (HNB) bromide and 2-nitrophenylsulfenyl chloride. Although the modified toxins almost completely lost their lethal toxicity, the antigenic specificity remained virtually unchanged. Each modified derivative gave a single fused precipitin line with cobrotoxin on immunodiffusion against either anti-cobrotoxin or anti-HNB-oobrotoxin sera . In heterologous precipitin reactions, no significant decreases in antigenic activity of the modified derivatives were observed when they reacted with anticobrotoxin sera . Among the modified toxins, HNB-cobrotoxin is the most superior to native toxin for inducing the production of antibody in animals. In fact, l ß times more potent antisera were obtained in only one-half the time by using HNB-cobrotoxin instead of native toxin. These results suggest that although the Trp-residue in cobrotoxin is essential for lethal toxicity, it may not be involved in the antigenic specificity of the toxin. C.C .Y.
Crtxxa, C. C., Ct~tv, T. F. and I-~, C. Y. (Pharmacol . Inst ., College of Medicine, National Taiwan Univ ., Taipei, Taiwan, Republic of China) . Studies of the presynaptic effect of ßbungarotoxin on neuromuscular transmission . J. Pharmac. exp. 79~er. 184, 339 (1973) .
Tt~ st ocl~a effect of ß-bungarotoxin (ß-BuTX) isolated from the venom of Bungarur multicixctus on neuromuscular transmission in the rat isolated diaphragm was studied. No postsynaptic action on the membrane potential, action potential or the sensitivity to acetykholine of the motor end-plate was observed . Since conduction in the phrenic nerve and its terminal also was unaffected, the action of ~BuTX seems to be confined exclusively to the presynaptic site . Both Ca'+-deficient and high Mg'+ media antagonized the 7YIXICON 1973 Yot. 11
binding as well as the change after binding which leads to the inhibitory effect . ß-BuTX did not reduce the store of actylcholine in the diaphragm even after complete neuromuscular blockade when the quantal content of end-plate potentials was markedly reduced. After neuromuscular blockade, bursts of miniature end-plate potentials could be evoked by hypertonicity or high K+ . It is concluded that the depression of the release of neurotransmitter by ß-BuTX is not due to depletion of the store, as previously postulated, but is due to an inhibition of the mechanism for releasing neurotransmitter . C.Y .
A. M., Fxmnucxs, K. K., Wnct~x, F. W. and Pxt~sco'rr, J. M. (Dept. Biochemistry and Biophysis, Texas A and M University, College Station, Texas, U.S .A .). Leucostoma Peptidase A: a metalloprotease from snake venom. Bicehim . biophys . Acta 293, 464, 1973 . SrlEx~f~v,
A PROTEOLYTIC enzyme (`Leucostoma Peptidase A') free of esterase and arylamidase activities was isolated from the venom of Agkistrodon pisclvorus leucostoma by ion exchange and gel chromatography. The enzyme has a pH optimum of pH 8~5 (substrate : hemoglobin) and a mol weight of 22,500. It hydrolyzes the polyaminoacid-derivatives of lysine, tyrosine, phenylalanine and tryptophane, several N-substituted dipeptides and the oxidised B-chain of insulin. The enzyme is not inhibited by serine specific reagents, but sensitive to o-phenanthroline and F.DTA . It is a metalloprotein containing two Ca'+ and one Zn'+ per molecule. All these characteristics-perhaps with exception of the molecular weight-indicate a similarity to metal chelator sensitive neutral professes, which so far have been isolated only from micro-organisms . H.M .
Rose, W. C., Btuot~r, S. G. and Lt:E, I. P. (Virginia Commonwealth University, Department of Microbiology, Richmond, Virginia, U.S .A .) . Potentiation of the toxicity of severalantitumor agents by SylmorttRa typhosa endotoxin. Toxic. appl . Pharmac. 23, 102, 1972 . i.n l or S. typhl endotoxin was combined with several antitumor agents. BALH/c mice were used and the administration was intraperitoneal. Lethality was observed up to one week . The endotoxin enhanced signi&antly the lethality of cyclophosphamide, daunomycin, methotrexate, polyI:C, procarbazine and vincristine. As man is very susceptible to endotoxin, the possible importance of these findings in man treated with antittmtor drugs iß discussed. H. R. AN
OXICON 1973 Yot. II
TsE ruxtFlcwnoty of a phosphodiesterase from Bothrops atrox venom by chromatography on phosphocellulose and affinity chromatography with O-(4-nitrophenyl}O'-phenyl-thiophosphate ester coupled to activated Sepharose is described. Phosphatases are removed by chromatography on hydroxyapatite . The enzyme is a single-chain protein with a molecular weight of approximately 130,01x1 as judged by dodecylsulfate-gel electrophoresis and ultracentrifugation. It has a specific activity of 3~3 pm bis (4-nitrophenyl) phosphate hydrolyzed min'' mg 1 at 24°C . W.F.K .
Toots, P. M. and Cxwta, D. S. (Dept. of Chemistry, University of Southern Mississippi, Hattiesburg, Mississippi 39401, U.S .A .). Enzymatic activities of venom from the jellyfish Stomolophus meleagris. Comp . Biochem. Physiol. 43B, 435, 1972 .
venom from the nematocysts of the jellyfish Stomolophus meleagris was tested for enzymatic activity on 28 substrates commonly hydrolyzed by many venoms. Twelve of the substrates tested were hydrolyzed, suggesting the presence of 5'-nucleotidase, hyaluronidase, acid and alkaline phosphatases, phosphodiesterase, leucine aminopeptidase and professes, all of which have also been found in snake venoms . On the other hand, a number of enzymes found in some snake venoms (e.g . acetylcholine esterase, blood clotting enzymes, amino acid oxidases, amino acid esterases, and phospholipases) were not detectod in venom of S. meleagris. The enzymatic nature and physiological actions of jellyfish venom are compared with those of snake venoms . P.R.S. LYOP~-t~~
Ct3.~,taa, C. C. and Y~tvct, C. C. (Dept. of Biochem., Kaohsiung Medical College, Kaohsiung, Taiwan, Republic of China) . Tmmunochemical studies on the tryptophan-modified cobrotoxin . Blochim. biophys. Acta 295, 595, 1973).
Mosr sruxE neurotoxins whose amino acid sequences have been established contain only one Trp-residue located at the homologous position. The single Trp-residue in cobrotoxin, a neurotoxic protein obtained from the venom of Taiwan cobra (NgJa ngja atra), has been converted into N-formylkynurenine by ozonization in formic acid, oxidised to oxindole derivative with N-bromosuocinimide, and also modified by reactions with 2-hydroxy-5-nitrobenzyl (HNB) bromide and 2-nitrophenylsulfenyl chloride. Although the modified toxins almost completely lost their lethal toxicity, the antigenic specificity remained virtually unchanged. Each modified derivative gave a single fused precipitin line with cobrotoxin on immunodiffusion against either anti-cobrotoxin or anti-HNB-oobrotoxin sera . In heterologous precipitin reactions, no significant decreases in antigenic activity of the modified derivatives were observed when they reacted with anticobrotoxin sera . Among the modified toxins, HNB-cobrotoxin is the most superior to native toxin for inducing the production of antibody in animals. In fact, l ß times more potent antisera were obtained in only one-half the time by using HNB-cobrotoxin instead of native toxin. These results suggest that although the Trp-residue in cobrotoxin is essential for lethal toxicity, it may not be involved in the antigenic specificity of the toxin. C.C .Y.
Crtxxa, C. C., Ct~tv, T. F. and I-~, C. Y. (Pharmacol . Inst ., College of Medicine, National Taiwan Univ ., Taipei, Taiwan, Republic of China) . Studies of the presynaptic effect of ßbungarotoxin on neuromuscular transmission . J. Pharmac. exp. 79~er. 184, 339 (1973) .
Tt~ st ocl~a effect of ß-bungarotoxin (ß-BuTX) isolated from the venom of Bungarur multicixctus on neuromuscular transmission in the rat isolated diaphragm was studied. No postsynaptic action on the membrane potential, action potential or the sensitivity to acetykholine of the motor end-plate was observed . Since conduction in the phrenic nerve and its terminal also was unaffected, the action of ~BuTX seems to be confined exclusively to the presynaptic site . Both Ca'+-deficient and high Mg'+ media antagonized the 7YIXICON 1973 Yot. 11
binding as well as the change after binding which leads to the inhibitory effect . ß-BuTX did not reduce the store of actylcholine in the diaphragm even after complete neuromuscular blockade when the quantal content of end-plate potentials was markedly reduced. After neuromuscular blockade, bursts of miniature end-plate potentials could be evoked by hypertonicity or high K+ . It is concluded that the depression of the release of neurotransmitter by ß-BuTX is not due to depletion of the store, as previously postulated, but is due to an inhibition of the mechanism for releasing neurotransmitter . C.Y .
A. M., Fxmnucxs, K. K., Wnct~x, F. W. and Pxt~sco'rr, J. M. (Dept. Biochemistry and Biophysis, Texas A and M University, College Station, Texas, U.S .A .). Leucostoma Peptidase A: a metalloprotease from snake venom. Bicehim . biophys . Acta 293, 464, 1973 . SrlEx~f~v,
A PROTEOLYTIC enzyme (`Leucostoma Peptidase A') free of esterase and arylamidase activities was isolated from the venom of Agkistrodon pisclvorus leucostoma by ion exchange and gel chromatography. The enzyme has a pH optimum of pH 8~5 (substrate : hemoglobin) and a mol weight of 22,500. It hydrolyzes the polyaminoacid-derivatives of lysine, tyrosine, phenylalanine and tryptophane, several N-substituted dipeptides and the oxidised B-chain of insulin. The enzyme is not inhibited by serine specific reagents, but sensitive to o-phenanthroline and F.DTA . It is a metalloprotein containing two Ca'+ and one Zn'+ per molecule. All these characteristics-perhaps with exception of the molecular weight-indicate a similarity to metal chelator sensitive neutral professes, which so far have been isolated only from micro-organisms . H.M .
Rose, W. C., Btuot~r, S. G. and Lt:E, I. P. (Virginia Commonwealth University, Department of Microbiology, Richmond, Virginia, U.S .A .) . Potentiation of the toxicity of severalantitumor agents by SylmorttRa typhosa endotoxin. Toxic. appl . Pharmac. 23, 102, 1972 . i.n l or S. typhl endotoxin was combined with several antitumor agents. BALH/c mice were used and the administration was intraperitoneal. Lethality was observed up to one week . The endotoxin enhanced signi&antly the lethality of cyclophosphamide, daunomycin, methotrexate, polyI:C, procarbazine and vincristine. As man is very susceptible to endotoxin, the possible importance of these findings in man treated with antittmtor drugs iß discussed. H. R. AN
OXICON 1973 Yot. II