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Proteolytic hemorrhagins in the venom of Bitis arietans: purification, characterization and substrate specificity
Eleventh World Congress
281
as well as at the various phases of vespine development (e.g. adults vs brood). Their presence in the alimentary canal as well as in storage tissues may play a role in defensive mechanisms. Annual variations o f toxicity in Venezuelan cattle prone to developing the bovine paraplegic syndrome: dependence on rain. C. Scvcik and G. D'Suze (Laboratory on Cellular Neuropharmacology, Centro de Biofisica y Bioquimica, Instituto Venezolano de Investigaciones Cientificas (IVIC), Apartado 21827, Caracas 1020, Venezuela). A clinical entity named bovine paraplegic syndrome (BPS) has spread in cattle growing areas of the central and eastern Venezuela. Approximately 4 million cattle are bred in the area where the disease occurs. The mortality ranges from 5 to 25% of the animals at risk, mostly pregnant or lactating cows. Characteristic of BPS is ventral or sternal decuaitus, in animals that make vain efforts to stand when stimulated. Death occurs after a few days, and there is no known treatment. Previously we showed the existence of a sodium channel-blocking toxin produced by ruminal bacteria, in cattle suffering, or liable to suffer from BPS. We have now followed the presence of this toxin in bovine plasma sampled monthly during the rainy and dry seasons in animals without BPS. We have found a positive correlation between rain precipitation and plasmatic toxin levels (Spearman's rank correlation coefficient = 0.457, n = 135, P < 10-~). The precipitation was 244.8 (171.9, 300) mm water/month (n = 6) (median and its 95% confidence interval) during the rainy season and 7.4 (0, 66.4) mm water/month (n = 5) during the dry season [P = 0.005, Mann-Whitney (Wicoxon) test]. The plasmatic toxicities determined by high performance liquid chromatography (Scvcik et al., 1993) expressed as peak units of area (PUA, 1 PUA = 2.5 x 10 -6 absorbance) at 340 nm were 21.7 (9.8, 41.1) PUA//al (n = 45) in the rainy season and 0.0 (0.0, 2.1) PUA/#I in the dry season (n = 88) (P = 3 x 10-6). The distribution of toxin concentration in both groups was also different (X2 test, P = 10 -s, 9 degrees of freedom); this determinated that toxin was not detectable in 77.8% of th,~ cows in the dry season (n = 108) and in 22.2% in the rainy season (n = 45). Since BPS has its maximum incidence during the rainy season (especially when it begins and ends), the results strengthen the hypothesis of ~: causative role of the toxin in BPS. Supported in p~rt by grants from the IFS, Sweden (No. B/2039-1, GD'S), PDVSA (No. 4.2, CS) and CONICIT, Venezuela (No SI-2086, CS); field support was by Agropecuaria Flora, branch of The Union International Ltd, U.K. Sevcik et al. (1993) Toxicon 31, 1581.
POSTERS Proteolytic hemorrhagin~ in the venom o f Bitis arietans: purification, characterization and substrate specificity. T. Omori-Satoh, I Y. Yamakawa, I Y. Nagaoka I and D. Mebs2 (t National Institute of Health, Tokyo, Japan; and 'Institut der Rechtsmedizin, Frankfurt, Germany). Two proteolytic hemorrhagins, BHRa and BHRb, were isolated from the venom of Bitis arietans. They were high tool. wt proteins with apparent mol. wts of 68 kD and 75 kD, respectively, being distinct immunologically. Susceptibility to various reagents suggested that they were not serine proteinases, but metalloproteinases. Both hemorrhagins hydrolysed type I, II, III and IV collagens in denatured forms, but only type IV collagen in native form. Time-dependent hydrolysis patterns of type 1V collagen in SDS-PAGE suggested that the hemorrhagins had different substrate specificity. The hemorrhagins did not hydrolyse synthetic substrates for bacterial and animal collagenases. Although they hydrolysed angiotensin l at the same position, they hydrolysed luteinizing hormone-relea,~ing hormone at different positions. The hemorrhagins had no exopeptidase activity. Substrate specificity of the hemorrhagins was further examined using insulin B-chain. Oxidized insulin B-chain was cleaved by BHRa in 10, by BHRb in l I positions. The time-dependent occurrence and disappearance of the peptides indicated that 1:heinitial cleavage by BHRa occurred at the Scrg-Hisj0, AlaL4-Lcujs, and Tyrl6-Leul7 bonds, and by BHRb at the Hisl0-Leual, Alal4-Leuls, Leui~-Tyrl6 and Tyri6-Leui7 bonds. The substrate specificity of the hemorrhagins was compared with those of hemorrhagic and nonhemorrhagic venom proteinases hitherto reported. Presence o f heat-stable hemorrhagic toxins in snake venoms. C. L. Ownby, T. R. Colberg and Q. Li (Department of Physiological Sciences, Oklahoma State University, Stillwater, OK 74078, U.S.A.). Twenty-eight snake venoms (seven Agki~trodon venoms, six Bothrops venoms, thirteen Crotalus venoms, one S/strurus venora, and one Bitis venom) were examined for the presence of heat-stable (100°C, 5 mini hemorrhagic toxins. Both heated and unheated venoms were analyzed for their protein composition by SDS--PAGE, and tested for their hemorrhagic activity in vivo in mice and proteolytic activity on two different substrates. Heating led
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as well as at the various phases of vespine development (e.g. adults vs brood). Their presence in the alimentary canal as well as in storage tissues may play a role in defensive mechanisms. Annual variations o f toxicity in Venezuelan cattle prone to developing the bovine paraplegic syndrome: dependence on rain. C. Scvcik and G. D'Suze (Laboratory on Cellular Neuropharmacology, Centro de Biofisica y Bioquimica, Instituto Venezolano de Investigaciones Cientificas (IVIC), Apartado 21827, Caracas 1020, Venezuela). A clinical entity named bovine paraplegic syndrome (BPS) has spread in cattle growing areas of the central and eastern Venezuela. Approximately 4 million cattle are bred in the area where the disease occurs. The mortality ranges from 5 to 25% of the animals at risk, mostly pregnant or lactating cows. Characteristic of BPS is ventral or sternal decuaitus, in animals that make vain efforts to stand when stimulated. Death occurs after a few days, and there is no known treatment. Previously we showed the existence of a sodium channel-blocking toxin produced by ruminal bacteria, in cattle suffering, or liable to suffer from BPS. We have now followed the presence of this toxin in bovine plasma sampled monthly during the rainy and dry seasons in animals without BPS. We have found a positive correlation between rain precipitation and plasmatic toxin levels (Spearman's rank correlation coefficient = 0.457, n = 135, P < 10-~). The precipitation was 244.8 (171.9, 300) mm water/month (n = 6) (median and its 95% confidence interval) during the rainy season and 7.4 (0, 66.4) mm water/month (n = 5) during the dry season [P = 0.005, Mann-Whitney (Wicoxon) test]. The plasmatic toxicities determined by high performance liquid chromatography (Scvcik et al., 1993) expressed as peak units of area (PUA, 1 PUA = 2.5 x 10 -6 absorbance) at 340 nm were 21.7 (9.8, 41.1) PUA//al (n = 45) in the rainy season and 0.0 (0.0, 2.1) PUA/#I in the dry season (n = 88) (P = 3 x 10-6). The distribution of toxin concentration in both groups was also different (X2 test, P = 10 -s, 9 degrees of freedom); this determinated that toxin was not detectable in 77.8% of th,~ cows in the dry season (n = 108) and in 22.2% in the rainy season (n = 45). Since BPS has its maximum incidence during the rainy season (especially when it begins and ends), the results strengthen the hypothesis of ~: causative role of the toxin in BPS. Supported in p~rt by grants from the IFS, Sweden (No. B/2039-1, GD'S), PDVSA (No. 4.2, CS) and CONICIT, Venezuela (No SI-2086, CS); field support was by Agropecuaria Flora, branch of The Union International Ltd, U.K. Sevcik et al. (1993) Toxicon 31, 1581.
POSTERS Proteolytic hemorrhagin~ in the venom o f Bitis arietans: purification, characterization and substrate specificity. T. Omori-Satoh, I Y. Yamakawa, I Y. Nagaoka I and D. Mebs2 (t National Institute of Health, Tokyo, Japan; and 'Institut der Rechtsmedizin, Frankfurt, Germany). Two proteolytic hemorrhagins, BHRa and BHRb, were isolated from the venom of Bitis arietans. They were high tool. wt proteins with apparent mol. wts of 68 kD and 75 kD, respectively, being distinct immunologically. Susceptibility to various reagents suggested that they were not serine proteinases, but metalloproteinases. Both hemorrhagins hydrolysed type I, II, III and IV collagens in denatured forms, but only type IV collagen in native form. Time-dependent hydrolysis patterns of type 1V collagen in SDS-PAGE suggested that the hemorrhagins had different substrate specificity. The hemorrhagins did not hydrolyse synthetic substrates for bacterial and animal collagenases. Although they hydrolysed angiotensin l at the same position, they hydrolysed luteinizing hormone-relea,~ing hormone at different positions. The hemorrhagins had no exopeptidase activity. Substrate specificity of the hemorrhagins was further examined using insulin B-chain. Oxidized insulin B-chain was cleaved by BHRa in 10, by BHRb in l I positions. The time-dependent occurrence and disappearance of the peptides indicated that 1:heinitial cleavage by BHRa occurred at the Scrg-Hisj0, AlaL4-Lcujs, and Tyrl6-Leul7 bonds, and by BHRb at the Hisl0-Leual, Alal4-Leuls, Leui~-Tyrl6 and Tyri6-Leui7 bonds. The substrate specificity of the hemorrhagins was compared with those of hemorrhagic and nonhemorrhagic venom proteinases hitherto reported. Presence o f heat-stable hemorrhagic toxins in snake venoms. C. L. Ownby, T. R. Colberg and Q. Li (Department of Physiological Sciences, Oklahoma State University, Stillwater, OK 74078, U.S.A.). Twenty-eight snake venoms (seven Agki~trodon venoms, six Bothrops venoms, thirteen Crotalus venoms, one S/strurus venora, and one Bitis venom) were examined for the presence of heat-stable (100°C, 5 mini hemorrhagic toxins. Both heated and unheated venoms were analyzed for their protein composition by SDS--PAGE, and tested for their hemorrhagic activity in vivo in mice and proteolytic activity on two different substrates. Heating led