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Snake proteins and nucleic acids
536
Tenth Europaaa Symposium
Trm v~osa from Crotalidoe and Yiperidoe soaks commonly produce local tissue damage . Local or extensive oedema, which repraenta the major sign of European viper envenoming, is due to marked increase in local vascular permeability. Many reports showed that local manifetations and especially oedema wen not prevented by the use of antivenom. This let us to examine whether low mol. wt peptides of weak antigenicity could be reponsible for the effect of the venom on capillary permeability. In this study, we examined : firstly, the affect of Yipero aapir venom and of its low ( < 6000) and high ( > 6000) mol. wt components on plasma extravasation in the sltin, capillary permeability increase (CPn being leafed on mice according to the method of Miles and Wilhehn; and secondly, the ability of increasing doses of antivenom (Fab'~ to inlribit this e&a. The reanlta showed that the high mol. wt oomponenta of the venom are responsible for its effect on CPI and that the low mol. wt peptides are sot. Low dose of antivenom (2.5 Irl or leas) effectivdy neutralized CPI when mixed in vitro with the venom (50pg/mice) prior to s.c. injecdon. A much Liner amount of antivenom (500 pl) was, however, neoaaary to inhibit CPI In vivo, when aativenom was i.v. injected bdore s.c. administration of the venom. Furthermore, when antivenom injection was done after venom administration, its effxtivenes was again significantly reduced. Our investigation showed that low mol. wt c~mpoaeats of Vipers aapit venom are not involved in the inflammatory menifeatations induced by this venom and confirmed the inefficacy of antivenoms to inhildt CPI itr vivo. The fact that antivenom is more active fir vitro than in vivo further might be related to the pharmaookinetica of tIu Fab'¢ of the antivenom. Investigations are in progress to tat the action of smaller immunoglobulin fragments.
Actfona ofjauicuifn-I mrd-2. antkholineaterau peptfdea isolatedJran green nrmnba venom, on end-plate cunenu at tire neuronmrtcwlar jrorctian. J. Mowo,' L. G. MAa"~ " nm ' J. M. Htmsen,,' P. JuzAra,' J. Smrx~ea and E.
Keruaaox' ('Laboratoire de Neurobiologie CelluLtire et Moléculaire, C.N .RS., 91198-Gif sur Yvette, Franse ; and =Department of Biochemistry, Biomedical Canter, University of UppsaLs, 5-75123 Uppsala, Sweden).
Fsxacurna (FAS) 1 and 2 are small peptide isoLrted from the venom of Derrdr»asptr mrgrottceps with a specific anticholinesterase action . They differ is their soquenoes by only one amino acid (KNt>sox et al., 1984, 1985). Preliminary X-ray analyses have bxn reported (iE Du et al., 1989; Ménez and Drxaeunc, 1990). We have analysed the effects of FASs on voltage-cLimped endpaaa of frog cutaneous pectoris muscla and comparod their actions with that of well-known aeetylcholirresteraee (AChE) inhibitors . FAS-1 (17-700nIv)) incxeaaed the amplitude ( as 40Yo) and the decay time constant s (300~OOYe) of normal (fast) miniature endplate currents (MEPC) resulting in an increase of the charge transferred of about five time. These effects were sustained and depended on the membrane potential. FAS-1 ailed on feat, slow and the ao-calkd'giant' MEPCs, increasing the relative proportion of the Lut two type . The giant MEPCs carried,a charge 200 lima Luger than that of normal MEPCa FAS-1 aLo transiently increased the peak amplitude and r of endplate ,currents (EPC) in junctions treated with (+hubocurariae ; however, this increase (200-300'/e) was Luger than for MEPCs. No marked differences ware detected between FAS-1 and FAS-2. FASs did not exhibit any of the channel-blocking actions produced by classical AChE inhibitors (carbamate derivative, neostigmine ; acridine derivatives: latrine, velnacrine; organophosphoroua compounds, armin and phospholine) even after a 10-20 times increase in concentration. These reaulta indicate that FASs are vary specific AChE inhibitors and could be useful in studying the influence of Luge ACh concentrations in the synaptic clap of cholinergic synapse. Axnmraox, A. J., Hertve~r, A. L. and MHUClUA, P. M. (1985) Nerrraret . Left . S, 123-128. Iüar~ox, E. P., Meuaue, P. M. and R~onruar~hnruwur~r~ D. (1984) I. Pkysfol., Paris 79, 232-240. ICeara9ox, E. P., Mauaue P. M. and itouruattez-Ixrrurea~t~e, D. (1985) PJmrmac. 7ber . 30, 259-276. Le Du, M. H., Mtitcrror, P., Houars, P. E. and FoxrECrr.rw-Ces~s, J. C. (1989) J. biol. Clrenr. 264, 21,401-21,402. Mi~ez R and DucAUnc, A. (1990) !. nrol. Biol. 216, 233-234. Supported in part by grants from Association Fraaçcaix contra lea Myopathies and a fellowship from Ministère de )a Rxhetche et de )a Technologie to L.G.M .
R Srbes;r.av,' P. Gouvr and A. Bsatacx' ('Département de Biochimie Médicale, Centre Médical Universitaire, 1 rue Michel Serval, 1211 Geneva 4, SwitzerLtnd; and =Fondation Culturelle Elapsoïdea, cana postale 98, 1219 Aire-Geneva, SwitzerLmd) . Snake protetnr and nucleic acids.
a complete list of all known amino-acid eequencea of proteins of venomous snakes, as well as a complete list of the corresponding published DNA sequences. All characterized proteins (including toxins, enzymes and non-toxic components from snake venoms, as well as proteins from other tissues, ouch as blood or muscle) are included with as much information as posdbk. In addition to the corresponding litereture refer+caca, the snake specie ad wall as the name of oath toxin or enzyme are given with both the common and scientific name, according to the latest nomenclatures in these fields. Nearly 400 sequence of proteins and nucleic acids WE rrevecompiLsd
Tenth European Symposium
537
isolated from veaomow snakes, mainly of biologically active components, have been obtained using the SWISS-PROT protein aequena database (B~ocx and HoOCt~urnv, 1992), the AZEMIOPS database oa venomous snakes (Gour and STbcttt.ttv, 1991) and the literature in the flekl as main wuroes . Information such as function, catalytic activity, location of disulfide bridges, thra~dimensional structure, subunits, venom source, mol. wt ~o , functional groups, and glyooaylation sites are also given. This should allow, in the near future, the optimization of the chuusiflcation of snake venom components, as well as the classification of the snakes themselves in the evolutionary context.
Merebnmee effectr ojergeeüeatoxiee IL D. ~tJPUT and M. Btn1c (Inatituu of Pathophysiology, School of Medicine, University Ljubljana, 61105 Ljubljana, Slovenia). BQua+~~+oxnv II (EqT m is the most abundant hemolytic and cardiotoxic polypeptide isolated from the sea anemo~ Actbefa equine L. It is analogous to the equinatoxin (EqT) descibed previously by Fatt,ex and Leeez (1974) . In rat it causes cardiac arrgt, coronary vasoapaam and other dose-dependent cardiac effects. In atria it induces transient negative followed by positive inotropic effects, but in ventricles it causes a strong negative inotropic effect only. Aa cardiotoxic effects of EqT have been usually attributed to the coronary vaeospaam it seemed reasonable to investigate the vasoconstriction in more detail. The vasoconstriction could be caused either by intravascular hemolytic and consequent hyperkelemia, or by diroct effects of EqT II on the smooth muscle membrane . In both ~ the membrane would become more permeable to calcium ions, which would cause contraction of smooth muscles. Direct membrane effects were studied by mesas of the 'patch clamp' method on erythrocytes and oa vascular smooth muscle cells. 'Vaseline gap voltage clamp' method was employed for measurements of membrane oonductsnoes in myelineted nerves and on single skeletal muscle fibres. Intravascular hemolytis was studied by measurements of hematocrit and spectrophotometrically. The ooatraction of vascular smooth muscle was measured both directly on pig coronary arteries and on rat aorta using mechano~lectrical transducers and indirectly by monitoring the perfu~on rate in the investigated vascular bed. Results revealed several dose-0ependent membrane effects of EqT II . The block of sodium currents in skeletal muscle and the block of voltage-dependent potassium currents in myelinated nerve fibres were described before. Those results were obtained with low concentrations of EqT II (leas than 10 nM for nerve and z I pM for the skeletal musde) . Results of the present study indicate the possibility that high concentrations of EqT II (100 nM-1 pM in myelinated nerve fibre and in smooth muscle, > 1 nM in skeletal muscles, and < 10 pM in erythrocytes) increase the unspecific membrane conduclence . Ensuing accumulation of calcium ions in the cytoplasm might explain the muscle contractions and the cell death when high doses of EqT II are used. However, measurements of the force of contraction of the coronary smooth muscle and aortic rings show no effect of EqT II even in 1 pM doses. This argues against the direct effects of EqT II on the vascular smooth muscle . Nevertheless, the perfusion of isolated organs fn vitro sad In vivo drops suddenly and dramatically after perfusion with EqT II . In the guinea-pig heart the coronary flow falls to less than 10% of the control value within the fast minute of perfusion with 1 nM EqT II . This might be explained either by the possibility that the toxin acts directly on the arterioLu sphincters or venules, or by liberation of vasoconstrictory subataaas from endothelium. Hemolysis may play an important role fn vivo . Potassium ions liberated from erythrocytes may cause vasoconstrictioa. Patch~;lamp experiments reveahxi that 1-10 pM EqT II activates calcium-dependent potassium channels in the erythrocyte as well as in the smooth muscle all membrane, regardless of the fact that both cell types contain different types of the calcium-activated potassium channels. As it is very unlikely that the intracellular concentration of calcium ions has changed in our experimental conditions, we suggest a more direct action of the toxin on the caktium-dependent potassium ehannela . Liberation of potassium ions from erythrocytes is in agreement with our results. However, further studies are necessary to explain the difference between the vasoconstrictory action of EqT II and the lack of its action on the isolated smooth muscle.
Effects of storeefieb (Synenceia trachynis) venom are eenaim mati jrog xturonrueculmJunctions. A. S. K.en~,' J.
Motoo,r J. X. Co>~.t.n,~ B. Hetv~oiv' and S. Trash ('The Musculoakeletal Sciences Research Institute, 2(90 Fox Mill Rd, Heradon, VA 22071, U.S.A.; rLaboratoire de Neurobiologie Cellulaire et Moléculaire, C.N .R.S ., 91198 Gif sur Yvette Cedex, France; ~Universitat de Barcelona, Fecultad de Medicine de Lleida, Dept Ciénciea Médiques Basiques, Lleida, Spain; and 'Department of Pharmacology, University of Lund, Sülvegatan 10, S-22362 Lund, Sweden). Tta? ~ toxicity of stoneflsh (Synmeala trachynis) venom was characterized by elxtrophysiological andelectron microscopic eta,n+na tjon of isolated marine and frog nerve-skeletal muscle proparations exposed to various ooncentratiom of venom. Low concentration of venom (2 .5-5 pg/ml) acted prasynaptically. They caused neurotransmitter depletion by eliciting transmitter release and inhibiting synaptic vesicle recycling. The rapoase was Na+ channel-independent (resistant to tetrodotoxin), required the presence of either Cap+ or Mgr+,
Tenth Europaaa Symposium
Trm v~osa from Crotalidoe and Yiperidoe soaks commonly produce local tissue damage . Local or extensive oedema, which repraenta the major sign of European viper envenoming, is due to marked increase in local vascular permeability. Many reports showed that local manifetations and especially oedema wen not prevented by the use of antivenom. This let us to examine whether low mol. wt peptides of weak antigenicity could be reponsible for the effect of the venom on capillary permeability. In this study, we examined : firstly, the affect of Yipero aapir venom and of its low ( < 6000) and high ( > 6000) mol. wt components on plasma extravasation in the sltin, capillary permeability increase (CPn being leafed on mice according to the method of Miles and Wilhehn; and secondly, the ability of increasing doses of antivenom (Fab'~ to inlribit this e&a. The reanlta showed that the high mol. wt oomponenta of the venom are responsible for its effect on CPI and that the low mol. wt peptides are sot. Low dose of antivenom (2.5 Irl or leas) effectivdy neutralized CPI when mixed in vitro with the venom (50pg/mice) prior to s.c. injecdon. A much Liner amount of antivenom (500 pl) was, however, neoaaary to inhibit CPI In vivo, when aativenom was i.v. injected bdore s.c. administration of the venom. Furthermore, when antivenom injection was done after venom administration, its effxtivenes was again significantly reduced. Our investigation showed that low mol. wt c~mpoaeats of Vipers aapit venom are not involved in the inflammatory menifeatations induced by this venom and confirmed the inefficacy of antivenoms to inhildt CPI itr vivo. The fact that antivenom is more active fir vitro than in vivo further might be related to the pharmaookinetica of tIu Fab'¢ of the antivenom. Investigations are in progress to tat the action of smaller immunoglobulin fragments.
Actfona ofjauicuifn-I mrd-2. antkholineaterau peptfdea isolatedJran green nrmnba venom, on end-plate cunenu at tire neuronmrtcwlar jrorctian. J. Mowo,' L. G. MAa"~ " nm ' J. M. Htmsen,,' P. JuzAra,' J. Smrx~ea and E.
Keruaaox' ('Laboratoire de Neurobiologie CelluLtire et Moléculaire, C.N .RS., 91198-Gif sur Yvette, Franse ; and =Department of Biochemistry, Biomedical Canter, University of UppsaLs, 5-75123 Uppsala, Sweden).
Fsxacurna (FAS) 1 and 2 are small peptide isoLrted from the venom of Derrdr»asptr mrgrottceps with a specific anticholinesterase action . They differ is their soquenoes by only one amino acid (KNt>sox et al., 1984, 1985). Preliminary X-ray analyses have bxn reported (iE Du et al., 1989; Ménez and Drxaeunc, 1990). We have analysed the effects of FASs on voltage-cLimped endpaaa of frog cutaneous pectoris muscla and comparod their actions with that of well-known aeetylcholirresteraee (AChE) inhibitors . FAS-1 (17-700nIv)) incxeaaed the amplitude ( as 40Yo) and the decay time constant s (300~OOYe) of normal (fast) miniature endplate currents (MEPC) resulting in an increase of the charge transferred of about five time. These effects were sustained and depended on the membrane potential. FAS-1 ailed on feat, slow and the ao-calkd'giant' MEPCs, increasing the relative proportion of the Lut two type . The giant MEPCs carried,a charge 200 lima Luger than that of normal MEPCa FAS-1 aLo transiently increased the peak amplitude and r of endplate ,currents (EPC) in junctions treated with (+hubocurariae ; however, this increase (200-300'/e) was Luger than for MEPCs. No marked differences ware detected between FAS-1 and FAS-2. FASs did not exhibit any of the channel-blocking actions produced by classical AChE inhibitors (carbamate derivative, neostigmine ; acridine derivatives: latrine, velnacrine; organophosphoroua compounds, armin and phospholine) even after a 10-20 times increase in concentration. These reaulta indicate that FASs are vary specific AChE inhibitors and could be useful in studying the influence of Luge ACh concentrations in the synaptic clap of cholinergic synapse. Axnmraox, A. J., Hertve~r, A. L. and MHUClUA, P. M. (1985) Nerrraret . Left . S, 123-128. Iüar~ox, E. P., Meuaue, P. M. and R~onruar~hnruwur~r~ D. (1984) I. Pkysfol., Paris 79, 232-240. ICeara9ox, E. P., Mauaue P. M. and itouruattez-Ixrrurea~t~e, D. (1985) PJmrmac. 7ber . 30, 259-276. Le Du, M. H., Mtitcrror, P., Houars, P. E. and FoxrECrr.rw-Ces~s, J. C. (1989) J. biol. Clrenr. 264, 21,401-21,402. Mi~ez R and DucAUnc, A. (1990) !. nrol. Biol. 216, 233-234. Supported in part by grants from Association Fraaçcaix contra lea Myopathies and a fellowship from Ministère de )a Rxhetche et de )a Technologie to L.G.M .
R Srbes;r.av,' P. Gouvr and A. Bsatacx' ('Département de Biochimie Médicale, Centre Médical Universitaire, 1 rue Michel Serval, 1211 Geneva 4, SwitzerLtnd; and =Fondation Culturelle Elapsoïdea, cana postale 98, 1219 Aire-Geneva, SwitzerLmd) . Snake protetnr and nucleic acids.
a complete list of all known amino-acid eequencea of proteins of venomous snakes, as well as a complete list of the corresponding published DNA sequences. All characterized proteins (including toxins, enzymes and non-toxic components from snake venoms, as well as proteins from other tissues, ouch as blood or muscle) are included with as much information as posdbk. In addition to the corresponding litereture refer+caca, the snake specie ad wall as the name of oath toxin or enzyme are given with both the common and scientific name, according to the latest nomenclatures in these fields. Nearly 400 sequence of proteins and nucleic acids WE rrevecompiLsd
Tenth European Symposium
537
isolated from veaomow snakes, mainly of biologically active components, have been obtained using the SWISS-PROT protein aequena database (B~ocx and HoOCt~urnv, 1992), the AZEMIOPS database oa venomous snakes (Gour and STbcttt.ttv, 1991) and the literature in the flekl as main wuroes . Information such as function, catalytic activity, location of disulfide bridges, thra~dimensional structure, subunits, venom source, mol. wt ~o , functional groups, and glyooaylation sites are also given. This should allow, in the near future, the optimization of the chuusiflcation of snake venom components, as well as the classification of the snakes themselves in the evolutionary context.
Merebnmee effectr ojergeeüeatoxiee IL D. ~tJPUT and M. Btn1c (Inatituu of Pathophysiology, School of Medicine, University Ljubljana, 61105 Ljubljana, Slovenia). BQua+~~+oxnv II (EqT m is the most abundant hemolytic and cardiotoxic polypeptide isolated from the sea anemo~ Actbefa equine L. It is analogous to the equinatoxin (EqT) descibed previously by Fatt,ex and Leeez (1974) . In rat it causes cardiac arrgt, coronary vasoapaam and other dose-dependent cardiac effects. In atria it induces transient negative followed by positive inotropic effects, but in ventricles it causes a strong negative inotropic effect only. Aa cardiotoxic effects of EqT have been usually attributed to the coronary vaeospaam it seemed reasonable to investigate the vasoconstriction in more detail. The vasoconstriction could be caused either by intravascular hemolytic and consequent hyperkelemia, or by diroct effects of EqT II on the smooth muscle membrane . In both ~ the membrane would become more permeable to calcium ions, which would cause contraction of smooth muscles. Direct membrane effects were studied by mesas of the 'patch clamp' method on erythrocytes and oa vascular smooth muscle cells. 'Vaseline gap voltage clamp' method was employed for measurements of membrane oonductsnoes in myelineted nerves and on single skeletal muscle fibres. Intravascular hemolytis was studied by measurements of hematocrit and spectrophotometrically. The ooatraction of vascular smooth muscle was measured both directly on pig coronary arteries and on rat aorta using mechano~lectrical transducers and indirectly by monitoring the perfu~on rate in the investigated vascular bed. Results revealed several dose-0ependent membrane effects of EqT II . The block of sodium currents in skeletal muscle and the block of voltage-dependent potassium currents in myelinated nerve fibres were described before. Those results were obtained with low concentrations of EqT II (leas than 10 nM for nerve and z I pM for the skeletal musde) . Results of the present study indicate the possibility that high concentrations of EqT II (100 nM-1 pM in myelinated nerve fibre and in smooth muscle, > 1 nM in skeletal muscles, and < 10 pM in erythrocytes) increase the unspecific membrane conduclence . Ensuing accumulation of calcium ions in the cytoplasm might explain the muscle contractions and the cell death when high doses of EqT II are used. However, measurements of the force of contraction of the coronary smooth muscle and aortic rings show no effect of EqT II even in 1 pM doses. This argues against the direct effects of EqT II on the vascular smooth muscle . Nevertheless, the perfusion of isolated organs fn vitro sad In vivo drops suddenly and dramatically after perfusion with EqT II . In the guinea-pig heart the coronary flow falls to less than 10% of the control value within the fast minute of perfusion with 1 nM EqT II . This might be explained either by the possibility that the toxin acts directly on the arterioLu sphincters or venules, or by liberation of vasoconstrictory subataaas from endothelium. Hemolysis may play an important role fn vivo . Potassium ions liberated from erythrocytes may cause vasoconstrictioa. Patch~;lamp experiments reveahxi that 1-10 pM EqT II activates calcium-dependent potassium channels in the erythrocyte as well as in the smooth muscle all membrane, regardless of the fact that both cell types contain different types of the calcium-activated potassium channels. As it is very unlikely that the intracellular concentration of calcium ions has changed in our experimental conditions, we suggest a more direct action of the toxin on the caktium-dependent potassium ehannela . Liberation of potassium ions from erythrocytes is in agreement with our results. However, further studies are necessary to explain the difference between the vasoconstrictory action of EqT II and the lack of its action on the isolated smooth muscle.
Effects of storeefieb (Synenceia trachynis) venom are eenaim mati jrog xturonrueculmJunctions. A. S. K.en~,' J.
Motoo,r J. X. Co>~.t.n,~ B. Hetv~oiv' and S. Trash ('The Musculoakeletal Sciences Research Institute, 2(90 Fox Mill Rd, Heradon, VA 22071, U.S.A.; rLaboratoire de Neurobiologie Cellulaire et Moléculaire, C.N .R.S ., 91198 Gif sur Yvette Cedex, France; ~Universitat de Barcelona, Fecultad de Medicine de Lleida, Dept Ciénciea Médiques Basiques, Lleida, Spain; and 'Department of Pharmacology, University of Lund, Sülvegatan 10, S-22362 Lund, Sweden). Tta? ~ toxicity of stoneflsh (Synmeala trachynis) venom was characterized by elxtrophysiological andelectron microscopic eta,n+na tjon of isolated marine and frog nerve-skeletal muscle proparations exposed to various ooncentratiom of venom. Low concentration of venom (2 .5-5 pg/ml) acted prasynaptically. They caused neurotransmitter depletion by eliciting transmitter release and inhibiting synaptic vesicle recycling. The rapoase was Na+ channel-independent (resistant to tetrodotoxin), required the presence of either Cap+ or Mgr+,