- Email: [email protected]
The unconventional amino acid sequence of the major neurotoxic polypeptide of the sun anemone Stoichactis helianthus
$80
8th World Coagre~ on Animal, Plant and Microbial Toxins
STUDYOF THE COAGULANTAND PROTEOLYTICACTIVITIESOF NEWBORN BOTHROPSJARARACAVENOM. A. S. KAMIGUTIl and S. HANAOA 2. Laboratory of Hematology of the Instituto ButantanI and Dept of Pharmacology of the Universidade de S~o Paulo~, S~o Paulo, Brazil. Venoms obtained from young and adult snakes have been reported to show different clotting activities ( l ) . Among~throps venoms, i t was recorded that venom from young ~th_~ ara~ was mere p o t e n ~ o t t i n g horse plasma than that from adult spec linens Lz). The present investigation aimed to explain the mode of action of newborn B. Jareraca venom upon blood c l o t t i n g factors, as well as to study i t s proteolytic ~'ctTvly~ompared to the venom of adult ones. I t was found that: a) The venom of newborn snakes has thrombin-like a c t i v i t y , which is as high as that of adult. Its procoagulant activity is due to activation of both factor X and prothrombin. Factor X activation is four times increased with newborn venom. The five minutes-incubated mixture of prothrombin and 2ug venom in the absence of factor V generated thrombin amount corresponding to 9 and 1.5 NIHu/ml, respectively with newborn and adult venoms. b) The pruteolytlc a c t i v i t y upon f i b r i n and azocoll is low, but on casein is higher than that of adult venom. REFERENCES (I) Reid, H. A. and Theakston, R. D. G. (1978) Am. J. Trop. Med. Hyg. 27, I053. (2) Rosenfeld, F., Hampe, O. G. and Kelen, E. M. A. (1959) Mere. Inst. Butantan29, 143. KEY WORDS Bothrops jararaca; venom of newborn snake; clotting and proteolytic activities.
THE UNCONVENTIONALAMINO ACID SEQUENCEOF THE MAJORNEUROTOXIC POLYPEPTIDE OF THE SUN ANEMONESTOICHACTISHELIANTHUS By W. R. KEM, B. H. DUNN, B. PARTEN, and D. PRICE. Depts. Of Pharmcolo(3), and Riochemistry, Universtl;y of Flortda College of Medicine, Gatnesvflle, FL 32610 and Whitney Marine Laboratory, Untverstl;y of Floride, St. Augustine, FL 32084, U.S.A. Stoichactts heltanthus possesses, tn addition to 17,000 dalton basic protein cytolyslns {Bernhelemr and Avtgud, 1976; alumenthal and Kern, 1983), two S,O00 dalton polypeptide neurotoxtns homelogous to the ftve polypeptides previously sequenced by several laboratories (see Scheffler et a l . , 1982). We have purified and sequenced the least basic toxin. Sh-NI was t s o l a - ~ freeze-thaw aqueous extraction from whole antmals, ammonium sulfate precipitation at 60Z saturation, G5O Sephadex, CM cellulose, and P cellulose chroemtography; the resulting toxin was homogeneous by PAGEand PAGZEF. I t was extreemly toxic to crayfish (LDBo about 0.6 pg/kg,3tntrahemecoeltc t~ect#on) but without effect upon mice at 15,000 ,g/kg ( t . p . ) . The H-RCMtoxin and i t s fragmnts were subjected to autoemttc Edmn degradation and the resulting PTH-a~dno acids were i d e n t i f i e d by HPLC, back hydrolyses, and s c i n t i l l a t i o n counting. A l l possible pepttdes resulting from clostrtpain digestion, staphylococcal I~otease digestion, or tr~ptophan pepttde cleavage (CNBr-HFBA) were tsolated and t h e i r amino actd compositions determined; a l l except two of these L)eptid~ns were also uenced. The sequence ts as f o l 35 40 4s~ 5 10 15 20 ~ o ~ v lows: AACKCDDEGPDIRTAPLTGTVDLGSCNAGkrEKCASYYTIIADCCRK.Only 281[ of this sequence is identical with the sequence (57% of residues) common to the other ftve toxins. All six half cystlnyl residues are in positions egutvalent to those tn the other toxins and thus account for about half of the toxin--homology" with the other toxins. The other identical residues are GLY 9, PRO 10, ARG 13, GLY 19, GLY 29, TRP 30, and ILE 40. We suggest that these restdues may he c r i t i c a l for polypepttde folding, or for making direct contact with the sodium channel. Supported by GM32848. REFERENCES Bernhetmer, A.W. and Avtgad, IS. (1976) Proc. Natl. Aced. Sct. USA 73, 467. Bluwenthal, K.M. and Kern, W.R. (1983) a. Biol. Chem. 257, 5574. Scheffler, a . - d . , Tsugtta, A., Linden, G., Schwettz, H. and Lazdunskt, M. (1982) Btochem. Btophys. Res. Comm. 107, 272. KEY WORDS Stotchactts helianthus; neuretoxin; structure
8th World Coagre~ on Animal, Plant and Microbial Toxins
STUDYOF THE COAGULANTAND PROTEOLYTICACTIVITIESOF NEWBORN BOTHROPSJARARACAVENOM. A. S. KAMIGUTIl and S. HANAOA 2. Laboratory of Hematology of the Instituto ButantanI and Dept of Pharmacology of the Universidade de S~o Paulo~, S~o Paulo, Brazil. Venoms obtained from young and adult snakes have been reported to show different clotting activities ( l ) . Among~throps venoms, i t was recorded that venom from young ~th_~ ara~ was mere p o t e n ~ o t t i n g horse plasma than that from adult spec linens Lz). The present investigation aimed to explain the mode of action of newborn B. Jareraca venom upon blood c l o t t i n g factors, as well as to study i t s proteolytic ~'ctTvly~ompared to the venom of adult ones. I t was found that: a) The venom of newborn snakes has thrombin-like a c t i v i t y , which is as high as that of adult. Its procoagulant activity is due to activation of both factor X and prothrombin. Factor X activation is four times increased with newborn venom. The five minutes-incubated mixture of prothrombin and 2ug venom in the absence of factor V generated thrombin amount corresponding to 9 and 1.5 NIHu/ml, respectively with newborn and adult venoms. b) The pruteolytlc a c t i v i t y upon f i b r i n and azocoll is low, but on casein is higher than that of adult venom. REFERENCES (I) Reid, H. A. and Theakston, R. D. G. (1978) Am. J. Trop. Med. Hyg. 27, I053. (2) Rosenfeld, F., Hampe, O. G. and Kelen, E. M. A. (1959) Mere. Inst. Butantan29, 143. KEY WORDS Bothrops jararaca; venom of newborn snake; clotting and proteolytic activities.
THE UNCONVENTIONALAMINO ACID SEQUENCEOF THE MAJORNEUROTOXIC POLYPEPTIDE OF THE SUN ANEMONESTOICHACTISHELIANTHUS By W. R. KEM, B. H. DUNN, B. PARTEN, and D. PRICE. Depts. Of Pharmcolo(3), and Riochemistry, Universtl;y of Flortda College of Medicine, Gatnesvflle, FL 32610 and Whitney Marine Laboratory, Untverstl;y of Floride, St. Augustine, FL 32084, U.S.A. Stoichactts heltanthus possesses, tn addition to 17,000 dalton basic protein cytolyslns {Bernhelemr and Avtgud, 1976; alumenthal and Kern, 1983), two S,O00 dalton polypeptide neurotoxtns homelogous to the ftve polypeptides previously sequenced by several laboratories (see Scheffler et a l . , 1982). We have purified and sequenced the least basic toxin. Sh-NI was t s o l a - ~ freeze-thaw aqueous extraction from whole antmals, ammonium sulfate precipitation at 60Z saturation, G5O Sephadex, CM cellulose, and P cellulose chroemtography; the resulting toxin was homogeneous by PAGEand PAGZEF. I t was extreemly toxic to crayfish (LDBo about 0.6 pg/kg,3tntrahemecoeltc t~ect#on) but without effect upon mice at 15,000 ,g/kg ( t . p . ) . The H-RCMtoxin and i t s fragmnts were subjected to autoemttc Edmn degradation and the resulting PTH-a~dno acids were i d e n t i f i e d by HPLC, back hydrolyses, and s c i n t i l l a t i o n counting. A l l possible pepttdes resulting from clostrtpain digestion, staphylococcal I~otease digestion, or tr~ptophan pepttde cleavage (CNBr-HFBA) were tsolated and t h e i r amino actd compositions determined; a l l except two of these L)eptid~ns were also uenced. The sequence ts as f o l 35 40 4s~ 5 10 15 20 ~ o ~ v lows: AACKCDDEGPDIRTAPLTGTVDLGSCNAGkrEKCASYYTIIADCCRK.Only 281[ of this sequence is identical with the sequence (57% of residues) common to the other ftve toxins. All six half cystlnyl residues are in positions egutvalent to those tn the other toxins and thus account for about half of the toxin--homology" with the other toxins. The other identical residues are GLY 9, PRO 10, ARG 13, GLY 19, GLY 29, TRP 30, and ILE 40. We suggest that these restdues may he c r i t i c a l for polypepttde folding, or for making direct contact with the sodium channel. Supported by GM32848. REFERENCES Bernhetmer, A.W. and Avtgad, IS. (1976) Proc. Natl. Aced. Sct. USA 73, 467. Bluwenthal, K.M. and Kern, W.R. (1983) a. Biol. Chem. 257, 5574. Scheffler, a . - d . , Tsugtta, A., Linden, G., Schwettz, H. and Lazdunskt, M. (1982) Btochem. Btophys. Res. Comm. 107, 272. KEY WORDS Stotchactts helianthus; neuretoxin; structure