Charlotte L. Ownby

Isolation of a myotoxin from Bothrops asper venom: partial characterization and action on skeletal muscle.

A myotoxic phospholipase has been isolated from Bothrops asper venom by ion-exchange chromatography on CM-Sephadex followed by gel filtration on Sephadex G-75. The toxin is a basic polypeptide with an estimated molecular weight of 10,700. It has both phospholipase A and indirect hemolytic activities, but is devoid of proteolytic, direct hemolytic and hemorrhagic effects. When injected i.m. into mice the toxin induces a rapid increase in plasma creatine kinase levels and a series of degenerative events in skeletal muscle which lead to myonecrosis. The toxin induces an increase in intracellular calcium levels and is able to hydrolyze muscle phospholipids in vivo. Pretreatment with the calcium antagonist verapamil failed to prevent the myotoxic activity. It is proposed that B. asper myotoxin causes cell injury by disrupting the integrity of skeletal muscle plasma membrane and that myotoxicity is at least partially due to the phospholipase A activity of the toxin.

Lysine 49 phospholipase A2 proteins.

The structures of several K49 PLA2 proteins have been determined and these differ as a group in several regions from the closely related D49 PLA2 enzymes. One outstanding difference is the presence of a high number of positively charged residues in the C-terminal region which combined with the overall high number of conserved lysine residues gives the molecule an interfacial adsorption surface which is highly positively charged compared to the opposite surface of the molecule. Although some nucleotide sequences have been reported, progress in obtaining active recombinant proteins has been slow. The K49 proteins exert several toxic activities, including myotoxicity, anticoagulation and edema formation. The most studied of these activities is myotoxicity. The myotoxicity induced by the K49 PLA2 proteins is histologically similar to that caused by the D49 PLA2 myotoxins, with some muscle fiber types possibly more sensitive than others. Whereas it is clear that the K49 PLA2 myotoxins lyse the plasma membrane of the affected muscle cell in vivo, the exact mechanism of this lysis is not known. Also, it is not known whether the toxin is internalized before, during or after the initial lysis or ever. The K49 PLA2 toxins lyse liposomes and cells in culture and in the latter, the PLA2 myotoxins exert at least two distinct mechanisms of action, neither of which is well-characterized. While the K49 PLA2 proteins are enzymatically inactive on artificial substrates, the toxins cause fatty acid production in cell cultures. Whether the fatty acid release is due to the enzymatic activity of the K49 PLA2 or stimulation of tissue lipases, is unknown. While there may be a role for fatty acid production in one mechanism of myotoxicity, a second mechanism appears to be independent of enzymatic activity. Although we are beginning to understand more about the structure of these toxins, we still know little about the precise mechanism by which they interact with the skeletal muscle cell in vivo.

Ability of wedelolactone, heparin, and para-bromophenacyl bromide to antagonize the myotoxic effects of two crotaline venoms and their PLA2 myotoxins

We examined the ability of wedelolactone, heparin and para-bromophenacyl bromide to antagonize the myotoxic activity in mice of venoms from Crotalus viridis viridis and Agkistrodon contortrix laticinctus and two phospholipase A2 myotoxins, CVV myotoxin and ACL myotoxin, isolated from them. Myotoxicity was measured by the increase in plasma creatine kinase (CK) activity at two hours and histological changes in extensor digitorum longus muscle (EDL) at three hours after injection of the test solution. Both heparin and wedelolactone independently reduced the myotoxic effect of both crude venoms and both myotoxins, but wedelolactone was more effective. Wedelolactone plus heparin reduced the myotoxic effect of CVV myotoxin more than either antagonist alone. The PLA2 inhibitor, para-bromophenacyl bromide (pBPB), reduced the myotoxic effect of both myotoxins more than either wedelolactone or heparin. On the other hand, the myotoxic effect of polylysine was not reduced by either wedelolactone or para-bromophenacyl bromide, but it was reduced by heparin. These results indicate that wedelolactone, para-bromophenacyl bromide and heparin are antagonists of these two phospholipase A2 myotoxins, and that antagonism by the first two compounds may be due to a more specific interaction with these proteins than that by the latter.

Melittin and phospholipase A2 from bee (Apis mellifera) venom cause necrosis of murine skeletal muscle in vivo

Melittin and phospholipase A2 (PLA2) from bee (Apis mellifera) venom were rested for their ability to induce necrosis of skeletal muscle cells after intramuscular injection into mice. Light and electron microscopic examination of tissue indicated that both melittin (4 micrograms/g) and bee venom PLA2 (4 micrograms/g) caused necrosis of skeletal muscle cells within 30 min after i.m. injection. Early changes in the cells consisted of delta lesions, indicating a ruptured plasma membrane, and hypercontraction of myofibrils. By 24 hr the affected cells appeared as an amorphous mass of disorganized and disrupted myofibrils contained in an intact basal lamina. To ensure that the myotoxic activity of the melittin preparation was not due to contaminating. PLA2 activity, the preparation was treated with p-bromophenacyl bromide (p-BPB), a known inhibitor of PLA2 activity. The p-BPB-treated melittin was determined to have no detectable PLA2 activity using a sensitive muscle cell culture assay, and it still induced myonecrosis, although to a lesser extent and of a slower onset. Additionally, p-BPB treatment of purified bee venom PLA2 completely inhibited its myotoxic activity. These results indicate that both melittin and bee venom PLA2 are capable of inducing necrosis of skeletal muscle cells upon i.m. injection, and that the catalytic and myotoxic activities of bee venom PLA2 are inihibited by p-BPB. Also, melittin and contaminating PLA2 in the melittin fraction may be acting synergistically to induce a stronger and more rapid myotoxic effect than occurs with either alone.

A new method for quantitating hemorrhage induced by rattlesnake venoms: ability of polyvalent antivenom to neutralize hemorrhagic activity

Polyvalent (Crotalidae) antivenin was tested for its ability to neutralize the hemorrhagic activity of two crotaline venoms when mixed with them prior to injection. Hemorrhage was measured by two methods. In the first method an intradermal injection of venom produced a hemorrhagic spot which was quantitated by measuring diameters. In the second method the amount of hemoglobin in a muscle extract was measured after i.m. injection of venom. The results show that both methods are useful for quantitating hemorrhage induced by Crotalus viridis viridis and Crotalus atrox venoms. Antivenin neutralized the hemorrhagic activity of 240 micrograms C. v. viridis venom and 120 micrograms C. atrox venom per 0.05 ml. The question remains, can antivenin neutralize this amount of venom when injected independently of venom.

Structure, Function and Biophysical Aspects of the Myotoxins from Snake Venoms

AbstractSnake venom myotoxins can be categorized into three types: small, basic polypeptides such as myotoxin a and crotamine; cardiotoxins from cobra venoms; and phospholipase A2, toxins such as crotoxin and notexin. All three types of myotoxins induce depolarization and contraction of skeletal muscle cells. However, the myonecrosis induced by the small, basic polypeptide myotoxins is different from that induced by the cardiotoxins and phospholipase A2 myotoxins in that the former do not appear to lyse the sarcolemma whereas the latter two types cause lysis of the sarcolemma which is of rapid onset. Molecular properties of the toxins are similar in that they are all highly basic proteins, and a large portion of their surface charge is positive Also, they all have considerable β-sheet structure which may be involved in interaction with the membrane The purpose of this review is to describe the structure and function of these myotoxins and to evaluate features they might share which could shed light on the...

Ability of antiserum to myotoxin a from prairie rattlesnake (Crotalus viridis viridis) venom to neutralize local myotoxicity and lethal effects of myotoxin a and homologous crude venom.

Abstract Antiserum to myotoxin f isolated from prairie rattlesnake ( Crotalus viridis viridis ) venom was tested for its ability to neutralize the local myotoxic and lethal activities of myotoxin a and of crude C. v. viridis venom. Wyeths polyvalent (Crotalidae) antivenin was also tested for its ability to neutralize the crude venom. Using a light microscopic method to quantitate myonecrosis, the effect of myotoxin a , i.e. vacuolation, could be distinguished from total myonecrosis induced by whole venom. The results indicate that anti-myotoxin a serum is more effective in neutralizing local myonecrosis, but polyvalent antivenin is more effective in neutralizing lethality when they are mixed with crude prairie rattlesnake venom prior to injection.

A Molecular Mechanism for Lys49-Phospholipase A2 Activity Based on Ligand-induced Conformational Change.

Agkistrodon contortrix laticinctus myotoxin is a Lys49-phospholipase A2 (EC 3.1.1.4) isolated from the venom of the serpent A. contortrix laticinctus (broad-banded copperhead). We present here three monomeric crystal structures of the myotoxin, obtained under different crystallization conditions. The three forms present notable structural differences and reveal that the presence of a ligand in the active site (naturally presumed to be a fatty acid) induces the exposure of a hydrophobic surface (the hydrophobic knuckle) toward the C terminus. The knuckle in A. contortrix laticinctus myotoxin involves the side chains of Phe121 and Phe124 and is a consequence of the formation of a canonical structure for the main chain within the region of residues 118–125. Comparison with other Lys49-phospholipase A2 myotoxins shows that although the knuckle is a generic structural motif common to all members of the family, it is not readily recognizable by simple sequence analyses. An activation mechanism is proposed that relates fatty acid retention at the active site to conformational changes within the C-terminal region, a part of the molecule that has long been associated with Ca2+-independent membrane damaging activity and myotoxicity. This provides, for the first time, a direct structural connection between the phospholipase “active site” and the C-terminal “myotoxic site,” justifying the otherwise enigmatic conservation of the residues of the former in supposedly catalytically inactive molecules.

Detection of myotoxin a-like proteins in various snake venoms

Ninety-five venom samples from eight snake genera (Agkistrodon, Bitis, Bothrops, Calloselasma, Crotalus, Sistrurus, Naja and Vipera) including venoms of Crotalus species of different geographical origin were assayed using immunodiffusion or an ELISA for the presence of the small basic protein, myotoxin alpha, known to cause muscle necrosis. Of the eight genera investigated, only Crotalus and Sistrurus venoms contained detectable amounts of myotoxin alpha-like proteins. The venoms of 13 out of 17 rattlesnake species investigated contained proteins immunologically similar to myotoxin alpha, including 12 Crotalus species and one Sistrurus species. The highest amounts were detected in venoms of C. exsul, C. viridis oreganus and C. v. viridis. Qualitative differences in the presence or absence of myotoxin alpha-like proteins were observed in the venoms of C. cerastes, C. horridus, C. lepidus, C. mitchelli, C. scutulatus, C. viridis and S. catenatus specimens of different geographic origin. The toxin was not detected in the venoms obtained from C. adamanteus, C. atrox, C. enyo or C. vegrandis specimens. The toxin appears to be widely distributed among rattlesnake species in the new world, but may vary qualitatively by geographical region in several species and subspecies.

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

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 pI 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.