Sphingomyelinase D activity of brown recluse spider (Loxosceles reclusa) venom as studied by 31P-NMR: effects on the time-course of sphingomyelin hydrolysis.

The time-course for the hydrolysis of the D linkage of chicken egg yolk sphingomyelin in a Triton X-100 mixed micelle and of lysophosphotidylcholine micelles, as catalyzed by brown recluse spider venom and brown recluse spider toxin, was followed by phosphorous-31 nuclear magnetic resonance spectroscopy. The overall rate of hydrolysis of sphingomyelin in mixed micelles was found to be an order of magnitude faster than the hydrolysis of lysophosphotidylcholine. Incorporation of lysophosphotidylcholine into mixed micelles with Triton X-100 inhibited the lipase activity of brown recluse spider venom and brown recluse spider venom toxin. The effects of increased rates of overall reaction were observed with increased temperature and also with decreased ionic strength. The presence of divalent calcium ions was found to be necessary for hydrolytic activity, but only in catalytic amounts (less than 1 mM).

Effect of hyperbaric oxygen on sphingomyelinase D activity of brown recluse spider (Loxosceles reclusa) venom as studied by 31P nuclear magnetic resonance spectroscopy.

Hyperbaric oxygen (HBO) has been reported by some to be therapeutic for necrotic lesions induced by the venom of the brown recluse spider, Loxosceles reclusa. Others have reported no efficacy for this treatment. In this study, the effect of high pressure oxygen on an enzymatic activity of the toxin of this venom is reported. The time course for the hydrolysis of the phosphocholine ester bond of chicken egg yolk sphingomyelin, as catalyzed by brown recluse spider venom (BRSV) and venom treated with extended HBO (12 hr at 10 atmospheres), was followed by phosphorus-31 nuclear magnetic resonance spectroscopy. The venom and HBO-pretreated venom demonstrated sphingomyelinase D activity. Phospholipase C activity was not detected. The sphingomyelinase D activity of BRSV in three separate experiments was not altered by HBO. The HBO-pretreated venom, in all cases, did not exhibit an altered time course in the overall hydrolysis of the D linkage of sphingomyelin.

Pathogenesis of hemorrhage induced by hemorrhagic proteinase IV from timber rattlesnake (Crotalus horridus horridus) venom.

The effects on the vascular system of a purified toxin, hemorrhagic proteinase IV, from Crotalus horridus horridus venom were studied with emphasis on the pathogenesis of hemorrhage. White mice were injected intramuscularly with sublethal doses of the hemorrhagic toxin, and tissue samples were obtained at 5 and 30 min, 3 and 24 hr after the injection. There was a good correlation between amount of toxin injected and amount of hemorrhage. Microscopically, extensive areas of hemorrhage around muscle and adipose cells were observed just 5 min after injection. At later time periods the changes were similar, but the hemorrhage was more extensive. Many vessels were plugged by aggregations of platelets. Electron microscopy showed that endothelial cells of capillaries were affected to various degrees. Some were swollen and had plasma membranes that formed large blebs; others were thin and disrupted. In vessels where the intercellular junctions could be observed, they were intact even when the endothelial cells were damaged or ruptured, indicating hemorrhage per rhexis instead of per diapedesis. Basal lamina were often disorganized or absent. Both intravascular and extravascular fibrin deposition were commonly observed. Hemorrhagic proteinase IV from C. h. horridus venom induces hemorrhage per rhexis and platelet aggregation within 5 min of intramuscular injection into mice, and marked fibrin deposition within 30 min of injection.

Potentiation of the toxicity of basic peptides from rattlesnake venoms by sodium acetate.

The potentiating effect of sodium acetate on the toxicity of crotamine from Crotalus durissus terrificus venom, E toxin from Crotalus horridus horridus venom, and myotoxin a from Crotalus viridus viridis venom was examined. Subcutaneous injection of 6.3 mg/kg body weight of either crotamine or E toxin in 0.6 ml of water or myotoxin a in 0.6 ml of 0.05 M Tris/0.1 M NaCl buffer, pH 9.0, failed to produce lethality in mice. Injection of either E toxin or crotamine at doses of 4.0 mg/kg in 0.6 ml of 20 mM phosphate, pH 7.2, containing 1 M sodium chloride also failed to produce lethality. However, when any of the toxins were injected in 0.4 ml of 1 M sodium acetate, pH 7.0, lethality was observed. LD50 values of 1.43 mg/kg for E toxin, 1.39 mg/kg for crotamine and 0.56 mg/kg for myotoxin a were determined under these conditions. Lethality was also observed when either sodium propionate or sodium butyrate was used as a carrier for E toxin. The effect of these two buffers on crotamine and myotoxin a was not examined. Injection of E toxin s.c. in water followed at various time intervals with i.p. injections of 1 M sodium acetate produced lethality, even when the acetate was injected up to 4 hr after the toxin challenge.

Resistance of a hemorrhagic proteinase from timber rattlesnake venom to proteolytic degradation.

The proteolytic activity of hemorrhagic proteinase IV isolated from timber rattlesnake (Crotalus horridus horridus) venom was resistant to inactivation by trypsin, pronase and the proteolytic IIt fraction isolated from timber rattlesnake venom. SDS-polyacrylamide gel electrophoresis of the hemorrhagin incubated alone and with the three proteinases revealed that the addition of trypsin or the IIt fraction caused little apparent degradation of the hemorrhagin, whether or not the samples were reduced prior to electrophoresis. SDS electrophoresis of the hemorrhagin after incubation with pronase revealed a single band of 28,000 apparent molecular weight (as compared to 52,000 for the original hemorrhagin) if the samples were not reduced prior to electrophoresis, and a single band of 17,000 if reduced. If the hemorrhagin was reduced and alkylated, it was much more susceptible to hydrolysis by all three proteinases.

Isotachophoretic and immunological analysis of venoms from sea snakes (Laticauda semifasciata) and brown recluse spiders (Loxosceles reclusa) of different morphology, locality, sex, and developmental stages.

Sea snake venom: The venom compositions of sea snakes, Laticauda semifasciata, with different scale patterns were analyzed by isotachophoresis. The comparison showed quantitative rather than qualitative differences. Similarly, L. semifasciata venoms of Philippine and Japanese origins differed only in the quantity of certain proteins. Spider venom: 3. Loxosceles reclusa venom apparatus extract is rich in neutral and acidic proteins but contains relatively small quantities of basic proteins. Differences in venom apparatus extract composition between nymph and adult (male or female) were detected by isotachophoresis. The extracts of male and female venom apparatus were very similar. Extracts of venom apparatus of spiders collected in locations separated by 100 miles were the same.

A thrombin-like enzyme from timber rattlesnake venom.

The procoagulant component has been purified from timber rattlesnake (Crotalus horridus horridus) venom by DEAE-cellulose ion-exchange chromatography followed by affinity chromatography on immobilized p-aminobenzamidine and a final DEAE-Sepharose chromatography. As obtained, the procoagulant gave a single band of Mr 29 500 +/- 2000 on SDS-polyacrylamide gel electrophoresis whether or not the sample was reduced prior to electrophoresis. Schiff's stain indicated the presence of some carbohydrate. The procoagulant showed one predominant and four minor bands on discontinuous gel electrophoresis. All caused fibrinogen solutions to clot. Treatment of the preparation with neuraminidase did not cause the minor bands to comigrate with the major band. Amino acid analysis revealed the presence of eight half-cystines, all of which were present as cystines in the intact molecule. The procoagulant has 13 tryptophans per molecule and an extinction coefficient for a 1% solution at 280 nm of 26.3. This venom procoagulant was found to induce clotting by catalyzing the hydrolysis of only the A fibrinopeptide from the A alpha-chain of fibrinogen. It was not inhibited by protein trypsin inhibitors, N-ethylmaleimide or dithiothreitol, but it was inactivated by phenylmethylsulfonyl fluoride, indicating an active-center serine. The procoagulant catalyzed the release of negligible acid-soluble peptides from bovine serum albumin, casein and hemoglobin.

Isolation and characterization of a hemorrhagic proteinase from timber rattlesnake venom.

A protein isolated from timber rattlesnake (Crotalus horridus horridus) venom by ion-exchange and high-pressure liquid chromatography is hemorrhage inducing and lethal to mice (LD50 of 10 micrograms/g of body weight). It is a Ca2+- and Zn2+-containing proteinase and has the ability to hydrolyze hide powder azure. Atomic absorption spectroscopy shows 2.5 Ca2+ and 1 Zn2+ per protein monomer. The proteinase activity is destroyed by incubation with disulfide-reducing agents and by dialysis against ethylenediaminetetraacetate. Coincident with the loss of proteinase activity is a corresponding loss of lethal and hemorrhagic activities, suggesting that all three are related. Attempts to replace the metals and restore activity have been unsuccessful. Amino acid analysis and isoelectric focusing reveal that this component is an acidic protein (pI = 5.1) containing about 20 disulfide bonds and 507 residues. Reduction of one disulfide bond per molecule decreases proteinase activity by 50% while reduction of eight disulfide bonds decreases activity by 80%. Loss of hemorrhagic activity parallels the decrease in proteinase activity.

Enzymatic and hemolytic properties of brown recluse spider (Loxosceles reclusa) toxin and extracts of venom apparatus, cephalothorax and abdomen.

Loxosceles reclusa venom apparatus extract, toxin, cephalothorax and abdomen extracts were tested for six activities. Protease, lipase, nonspecific hydrolase and direct hemolytic activities were found primarily in abdomen extracts while sphingomyelinase activity appeared predominantly in the toxin. Appreciable complement-mediated hemolysis was not observed.

The identity of bovine liver "nothing dehydrogenase".

An assay system was developed which allowed the partial purification of the substrate for beef liver nothing dehydrogenase. This was also found to be a substrate for lactate dehydrogenase (LDH). Using electrophoretic separations of purified LDH and beef liver extracts, nothing dehydrogenase was determined to be primarily the H4 isoenzyme of LDH. The nothing dehydrogenase phenomena was also observed with highly-purified, commercially-obtained LDH.

Characterization of a fibrinogenase from northern copperhead (Agkistrodon contortrix mokasen) venom.

One of the fractions obtained by the carboxymethylcellulose ion-exchange chromatography of northern copperhead (Agkistrodon contortrix mokasen) venom prevented the thrombin-induced clotting of fibrinogen by proteolytically degrading the fibrinogen. The active component has been further purified to apparent electrophoretic homogeneity by molecular sieve chromatography. Sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis indicated a molecular weight of 22 900 +/- 600 for the purified enzyme. In addition to its fibrinogenase activity, it catalyzed the hydrolysis of hide power azure and had an intraperitoneal LD50 value in mice of less than 5.1 microgram/g body weight. The enzyme rapidly destroyed fibrinogen's ability to form clots. Electrophoresis of fibrinogen which had been incubated only a few minutes with the fibrinogenase revealed the rapid disappearance of the alpha-chain and the appearance of lower molecular weight fragments. The neutral pH optimum and ethylenediamine-tetraacetic acid (EDTA) and dithiothreitol sensitivity indicated that this enzyme belonged to the class metalloproteinases. Atomic absorption studies have revealed one zinc atom per molecule of protein. The apoenzyme's activity was restored by incubation with ZnCl2.

Subspecific variations in Agkistrodon contortrix venoms.

1. Commercially available preparations of venoms of three subspecies of copperhead snake (Agkistrodon contortrix) were compared as to toxicity, enzymatic activities, effect on a nerve-muscle preparation and capacity to induce clotting of a fibrinogen solution or plasma. 2. Northern copperhead venom contained apparent neurotoxic activities that were not present in broadbanded copperhead venom and only partially present in southern copperhead venom. 3. Procoagulant activity was demonstrated in whole northern copperhead venom in the absence of exogenous calcium. Procoagulant activity was present in certain isolated fractions of southern and broadbanded copperhead venoms, but was not apparent in the whole venoms. 4. Differences were noted in the levels of enzyme activities and electrophoretic patterns of the three venoms.