Venom Of Agkistrodon Contortrix Laticinctus Research Articles

Effects of a myotoxic Asp49 phospholipase A 2 (ACL-I PLA 2) isolated from Agkistrodon contortrix laticinctus snake venom on water transport in the isolated toad urinary bladder

An Asp49 PLA 2 (ACL-I PLA 2) was purified from the venom of Agkistrodon contortrix laticinctus by gel filtration and cation-exchange chromatography. It has a relative molecular mass of 14,000, and its N-terminal sequence has more than 65% of identity with other snake venom PLA 2s. ACL-I PLA 2 injected into the Tibialis anterior muscle of rats and mice at doses of 0.3 and 1.6 mg/kg, respectively, induced muscle fiber necrosis, cellular infiltration and edema 3 and 48 h after injection. The effect of the purified enzyme on water permeability was tested in the isolated toad urinary bladder. Water flow through the membrane was measured gravimetrically in bag preparations of the bladder. ACL-I PLA 2 (20 nM) did not significantly alter the water permeability in the bladder preparations, whereas ACL myotoxin (ACLMT), a Lys49 PLA 2 isolated from the same venom, at similar concentration significantly increased (81%) the water permeability. However, both toxins inhibited the AVP-stimulated water permeability. These results strongly suggest that PLA 2 activity is not involved in the ACLMT effect on water transport and the effect of ACL-I PLA 2 myotoxin on membrane permeability is mediated by mechanisms that are different in comparison to ACLMT.

The effect of post-translational modifications on the hemorrhagic activity of snake venom metalloproteinases

Metalloproteinases (MPs) are Zn +-dependent endoproteolytic enzymes, abundant in crotalid and viperid snake venoms. Most snake venom metalloproteinases (svMPs) are active on extracellular matrix components and this effect is thought to result in bleeding as a consequence of the basement membrane disruption in capillaries. Jararhagin and ACLH are hemorrhagic svMPs from Bothrops jararaca and Agkistrodon contortrix laticinctus venom, respectively. Both enzymes demonstrate proteolytic activity on fibrinogen and fibronectin and jararhagin inhibits collagen-induced platelet aggregation in vitro. This work describes the expression, purification and successful refolding of the recombinant ACLH zymogen (rPRO-ACLH) as well as the catalytic domain of jararhagin (rCDJARA). The heterologous proteins were produced in E. coli, an in vivo expression system that does not make post-translational modifications. The recombinant refolded proteins did not show any hemorrhagic activity in mice skin, as well as the native deglycosylated jararhagin and ACLH. However, they preserved their proteolytic activity on fibrinogen and fibronectin. It seems that the hemorrhagic properties of these hemorrhagins are dependent on post-translational modifications, whereas their proteolytic activity is not dependent on such modifications.

Regeneration and change of muscle fiber types after injury induced by a hemorrhagic fraction isolated from Agkistrodon contortrix laticinctus venom

Tibialis anterior (TA) muscles of rats were evaluated 3h, 3 and 30days after intramuscular injection of ACL hemorrhagic toxin I (ACLHT-I, 5mg/kg), partially purified from the venom of Agkistrodon contortrix laticinctus. Contralateral muscles were injected with saline. Three hours after ACLHT-1 injection: presence of hemorrhagic areas and myonecrotic muscle fibers. Three days: injured muscles showed areas in regeneration, some regions with delay of regeneration and bundles of normal fibers. An increased TA muscle weight was found when compared with the contralateral (0.45±0.03g versus 0.36±0.04g, p=0.04). Thirty days: areas of regenerated muscle fibers presented splits and centralized nuclei. Some regions were replaced by connective tissue. All muscle fiber types were injured but only the incidence of type IIC increased (3.4±2.0% versus 0.2±0.2%, p=0.0005). Regenerated areas of muscles were exclusively composed by fiber types II and IIC. Regenerated muscles decreased the muscle weight (0.49±0.1g versus 0.66±0.05g, p=0.03). In conclusion, ACLHT-I: (a) caused hemorrhage and muscle fiber injury; (b) injured both fiber types I and II; (c) increased the incidence of fiber type IIC and; (d) some muscle regions were replaced by connective tissue.

Development of an enzyme-linked immunosorbent assay (ELISA) for identification of venoms from snakes in the Agkistrodon genus

Q. Li and C. L. Ownby. Development of an enzyme-linked immunosorbent assay (ELISA) for identification of venoms from snakes in the Agkistrodon genus. Toxicon 32, 1315–1325, 1994.—An enzyme-linked immunosorbent assay (ELISA) using a purified myotoxin from the venom of Agkistrodon contortrix laticinctus (broad-banded copperhead) as immunogen was developed for potential use in the identification of envenomation by snakes belonging to the genus Agkistrodon native to North America. The specificity of the assay was tested using a total of 43 venom samples from snakes of diverse geographic locations. Venom samples used for cross-reactivity determination represent eight snake families including 14 species from the genus Crotalus. The assay detected venom from all Agkistrodon species tested without significant cross-reactivity with other venoms except for samples from two species of Bothrops which do not occur naturally north of Southern Mexico. The detection limit of the assay was 2 ng/ml for homologous crude venom dissolved in normal human serum. The assay was highly accurate in correlating optical densities with venom concentrations ( r = 0.997). The presence of the antigen in experimental envenomations was readily detected by the assay at an i.m. injection dosage of 0.1 μg/g. This ELISA is a promising test for identification of envenomations by species of Agkistrodon found in most of North America. It can also be used to study the kinetics of the myotoxin in experimental envenomations.

Isolation of a myotoxin from the venom of Agkistrodon contortrix laticinctus (broad-banded copperhead) and pathogenesis of myonecrosis induced by it in mice

E. K. Johnson and C. L. Ownby. Isolation of a myotoxin from the venom of Agkistrodon contortrix laticinctus (broad-banded copperhead) and pathogenesis of myonecrosis induced by it in mice. Toxicon 31, 243–255, 1993.—A myotoxin was isolated from the venom of the broad-banded copperhead ( Agkistrodon contortrix laticinctus) by HPLC using anion and cation exchange chromatography. The toxin has a mol. wt of approximately 14,000 and has a p I greater than 9. It does not have phospholipase A activity, but does induce myonecrosis of skeletal muscle cells characterized by a hypercontraction of myofilaments. Electron microscopic analysis showed that the myotoxin appears to disrupt the sarcolemma of skeletal muscle cells. ACL myotoxin is very similar in mol. wt, amino acid composition, and myotoxic activity to myotoxins isolated from the venoms of Bothrops asper and Bothrops nummifer from Central America, suggesting that homologs of this toxin may be found in other crotaline snake venoms.

Isolation of a hemorrhagic toxin from the venom of Agkistrodon contortrix laticinctus (broad-banded copperhead) and pathogenesis of the hemorrhage induced by the toxin in mice

1. 1. A hemorrhagic toxin was isolated from the venom of Agkistrodon contortrix laticinctus (Broad-Banded Copperhead) by Sephacryl S-200 HR column chromatography followed by high performance chromatography on Waters DEAE 5PW and protein Pak 125 columns. 2. 2. Homogeneity was determined by the presence of a single band in acrylamide gel electrophoresis with silver staining. 3. 3. ACL hemorrhagic toxin I has a molecular weight of about 29,000, is slightly acidic, and is a metalloprotease with activity towards the substrates N, N-dimethylcasein and bovine fibrinogen. Although the toxin is able to hydrolyze fibrinogen in vitro, it does not possess any defibrinogenating activity in vivo whereas the crude venom does show this activity. It has similar cleavage specificities to other snake venom hemorrhagic toxins. 4. 4. ACL hemorrhagic toxin I causes hemorrhage of rapid onset, present within 5 min of intramuscular injection into mice, and the pathogenesis is one of hemorrhage per rhexis in which capillary endothelial cells are ruptured.

Characterization of a Nonproteolytic Arginine Ester-hydrolyzing Enzyme from Snake Venom

Abstract An enzyme that catalyzes the hydrolysis of N-benzoyl-l-arginine ethyl ester and p-toluenesulfonyl-l-arginine methyl ester has been isolated from the venom of Agkistrodon contortrix laticinctus (broadbanded copperhead) by means of DEAE-cellulose chromatography. A high degree of homogeneity is suggested by sedimentation velocity, gel filtration, polyacetate electrophoresis, and isoelectric focusing. The purified enzyme has a sedimentation coefficient of 2.7 S, a diffusion coefficient of 8.3 x 10-7 cm2 per sec, and a molecular weight of 30,000. The isoelectric point, as determined by means of isoelectric focusing, was found to be 9.1. Enzymatic assays showed the preparation to be specific for arginine esters. The Km values determined with N-benzoyl-l-arginine ethyl ester and p-toluenesulfonyl-l-arginine methyl ester are 1.17 x 10-4 and 1.49 x10-3, respectively. The enzyme was inhibited by diisopropyl fluorophosphate, phenylmethylsulfonyl fluoride, and N-bromosuccinimide. Optimal rates of hydrolysis were observed from pH 7.5 to pH 8.5.