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HPLC-purified opossum (Didelphis virginiana) serum and its neutralization effect on crotalid venoms
9th World Congress
79
HPLC-purified opossum (Didelphis virginiana) serum and its neutralization effect on crotalid venoms. JULIO G. SOTO, MARIA S. SA~rrA and JOHN C. PEREZ (Department of Biology, Texas A & I University, Kingsville, Texas 78363, U.S.A.).
NEUTRALIZATION of Crotalidae, Viperidae and Elapidae hemorrhagic activity by crude Virginia opossum (Didephis virginiana) serum has been reported (HuANG and PEREZ, 1980; SOTO et al., 1988). In this study high performance liquid chromatography was used to purify opossum antihemorrhagic factors and hemorrhagic factors in snake venoms. Crude opossum serum, 250 mg, were fractionated with a Pharmacia KI5/100 column packed with Sephadex G-200. The active fractions were pooled and further fractionated with a Waters semi-preparative and analytical anion exchange (Protein PAK DEAE 5 PW) columns. Five milligrams of Crotalus adamanteus, C. atrox and C. horridus venoms were partially fractionated in a Waters Protein PAK DEAE 5 PW anion exchange column. C. m. molossus venom, 5 mg, were partially purified with a Waters cation exchange (PAK SP 5PW) column. Hemorrhagic and proteolytic activities of venom fractions were tested. Eleetrophoresis under denaturing and non-denaturing conditions was used to check purity. The crude opossum serum and purified antihemorrhagic factors neutralized hemorrhagic activity of crude venoms and purified fractions. The three venoms separated by anion exchange chromatography had hemorrhage peaks with retention times greater than 14min. Hemorrhagic activity of C.m. molossus was detected throughout the HPLC cation profile. REFERENCES HUANG, S. Y. and PEB~Z, J. C. (1980) Toxicon 18, 421. SOTO, J. G., PEREZ, J. C. and MINTON, S. A. Toxicon 26, 875.
A basic phospholipase A 2 from Naja nigricollis crawshawii venom inhibits thrombin formation by the prothombinase complex. STEINGRIMURSTEFANSSON, R. MANJUNATHAKINI and HERBERT J. EVANS (Department of Biochemistry, Medical College of Virginia, Virginia Commonwealth University, Richmond, Virginia 23298, U.S.A.).
SEVERALphospholipases A2 (PLA2) from snake venoms possess anticoagulant activity. The mechanism of their anticoagulant activity is unclear. To determine the site in the coagulation cascade at which the strongly anticoagulant basic PLA2 enzyme from N. nigricollis crawshawii venom acts, we examined its inhibition of the coagulation of bovine plasma initiated by Russell's viper venom (RVV), thromboplastin and thrombin. The basic PLA 2 inhibits clot formation initiated by RVV and thromboplastin activation, but not by thrombin. Thus the PLA 2 appears to inhibit the conversion of prothrombin to thrombin by the prothrombinase complex. To examine this inhibition, the clotting factors comprising the prothrombinase complex (factors X, V and prothrombin) were isolated from bovine plasma and reconstituted on lipid membranes. This system activates prothrombin to thrombin, which can be assayed with a thrombin specific fluorometric substrate, when Factor X is activated with RVV and Ca 2÷. The basic PLA2 inhibits the activation of prothrombin in this system. The mechanism of this inhibition is being investigated.
Applications o f a sensitive, GC-ECD analysis for anatoxin-A. DOUGLAS K. STEVENS1 and ROBERT I. KRIEGER2 (1University of Idaho, WOI Regional Program in Veterinary Medical Education, Washington Animal Disease Diagnostic Laboratory and Pharmacology/Toxicology Program, Moscow, Idaho 83843; and 2Department of Food and Agriculture, Worker Health and Safety Unit, 1220 N Street, Sacramento, California 95814, U.S.A.).
ANATOXIN-a (ANTX-a), 2-acetyl-9-azabicyclo[4.2.1lnon-2-ene, is a potent nicotinic cholinergic alkaloid produced by some toxigenic strains of Anabaena cyanobacteria LDml. = 0.25 mg/kg, i.p. mouse. Ingestion of water containing these microorganisms has been responsible for death of livestock, pets and wildlife. Recently, this laboratory has developed a sensitive GC-ECD analysis of ANTX-a (detection limit = 5 ng) (SrrvENs and KRIEGER, 1988). We have applied this method to a variety of samples. We have detected and isolated a toxigenic strain of Anabaena circinalis from a lake with no history of toxicity and which previous bioassays have been negative. An increase in the ANTX-a content per mg of material has been observed in the past 2 years indicating the possibility of a future toxic bloom. We have also examined other 'non-toxic' strains, and strains known to produce other anatoxins and have not detected any ANTX-a, thus supporting the distinction between toxic and non-toxic strains. REFERENCE STEVENS, D. K. and KRIEGER, R. I. (1988) J. analyt. Toxicol. (in press).
79
HPLC-purified opossum (Didelphis virginiana) serum and its neutralization effect on crotalid venoms. JULIO G. SOTO, MARIA S. SA~rrA and JOHN C. PEREZ (Department of Biology, Texas A & I University, Kingsville, Texas 78363, U.S.A.).
NEUTRALIZATION of Crotalidae, Viperidae and Elapidae hemorrhagic activity by crude Virginia opossum (Didephis virginiana) serum has been reported (HuANG and PEREZ, 1980; SOTO et al., 1988). In this study high performance liquid chromatography was used to purify opossum antihemorrhagic factors and hemorrhagic factors in snake venoms. Crude opossum serum, 250 mg, were fractionated with a Pharmacia KI5/100 column packed with Sephadex G-200. The active fractions were pooled and further fractionated with a Waters semi-preparative and analytical anion exchange (Protein PAK DEAE 5 PW) columns. Five milligrams of Crotalus adamanteus, C. atrox and C. horridus venoms were partially fractionated in a Waters Protein PAK DEAE 5 PW anion exchange column. C. m. molossus venom, 5 mg, were partially purified with a Waters cation exchange (PAK SP 5PW) column. Hemorrhagic and proteolytic activities of venom fractions were tested. Eleetrophoresis under denaturing and non-denaturing conditions was used to check purity. The crude opossum serum and purified antihemorrhagic factors neutralized hemorrhagic activity of crude venoms and purified fractions. The three venoms separated by anion exchange chromatography had hemorrhage peaks with retention times greater than 14min. Hemorrhagic activity of C.m. molossus was detected throughout the HPLC cation profile. REFERENCES HUANG, S. Y. and PEB~Z, J. C. (1980) Toxicon 18, 421. SOTO, J. G., PEREZ, J. C. and MINTON, S. A. Toxicon 26, 875.
A basic phospholipase A 2 from Naja nigricollis crawshawii venom inhibits thrombin formation by the prothombinase complex. STEINGRIMURSTEFANSSON, R. MANJUNATHAKINI and HERBERT J. EVANS (Department of Biochemistry, Medical College of Virginia, Virginia Commonwealth University, Richmond, Virginia 23298, U.S.A.).
SEVERALphospholipases A2 (PLA2) from snake venoms possess anticoagulant activity. The mechanism of their anticoagulant activity is unclear. To determine the site in the coagulation cascade at which the strongly anticoagulant basic PLA2 enzyme from N. nigricollis crawshawii venom acts, we examined its inhibition of the coagulation of bovine plasma initiated by Russell's viper venom (RVV), thromboplastin and thrombin. The basic PLA 2 inhibits clot formation initiated by RVV and thromboplastin activation, but not by thrombin. Thus the PLA 2 appears to inhibit the conversion of prothrombin to thrombin by the prothrombinase complex. To examine this inhibition, the clotting factors comprising the prothrombinase complex (factors X, V and prothrombin) were isolated from bovine plasma and reconstituted on lipid membranes. This system activates prothrombin to thrombin, which can be assayed with a thrombin specific fluorometric substrate, when Factor X is activated with RVV and Ca 2÷. The basic PLA2 inhibits the activation of prothrombin in this system. The mechanism of this inhibition is being investigated.
Applications o f a sensitive, GC-ECD analysis for anatoxin-A. DOUGLAS K. STEVENS1 and ROBERT I. KRIEGER2 (1University of Idaho, WOI Regional Program in Veterinary Medical Education, Washington Animal Disease Diagnostic Laboratory and Pharmacology/Toxicology Program, Moscow, Idaho 83843; and 2Department of Food and Agriculture, Worker Health and Safety Unit, 1220 N Street, Sacramento, California 95814, U.S.A.).
ANATOXIN-a (ANTX-a), 2-acetyl-9-azabicyclo[4.2.1lnon-2-ene, is a potent nicotinic cholinergic alkaloid produced by some toxigenic strains of Anabaena cyanobacteria LDml. = 0.25 mg/kg, i.p. mouse. Ingestion of water containing these microorganisms has been responsible for death of livestock, pets and wildlife. Recently, this laboratory has developed a sensitive GC-ECD analysis of ANTX-a (detection limit = 5 ng) (SrrvENs and KRIEGER, 1988). We have applied this method to a variety of samples. We have detected and isolated a toxigenic strain of Anabaena circinalis from a lake with no history of toxicity and which previous bioassays have been negative. An increase in the ANTX-a content per mg of material has been observed in the past 2 years indicating the possibility of a future toxic bloom. We have also examined other 'non-toxic' strains, and strains known to produce other anatoxins and have not detected any ANTX-a, thus supporting the distinction between toxic and non-toxic strains. REFERENCE STEVENS, D. K. and KRIEGER, R. I. (1988) J. analyt. Toxicol. (in press).