Mojave rattlesnakes (Crotalus scutulatus scutulatus) lacking the acidic subunit DNA sequence lack Mojave toxin in their venom.

The venom composition of Mojave rattlesnakes (Crotalus scutulatus scutulatus) differs in that some individuals have Mojave toxin and others do not. In order to understand the genetic basis for this difference, genomic DNA samples from Mojave rattlesnakes collected in Arizona, New Mexico, and Texas were analyzed for the presence of DNA sequences that relate to the acidic (Mta) and basic (Mtb) subunits of this toxin. DNA samples were subjected to PCR to amplify nucleotide sequences from second to fourth exons of the acidic and basic subunits. These nucleotide sequences were cloned and sequenced. The nucleotide sequences generated aligned exactly to previously published nucleotide sequences of Mojave toxin. All DNA samples analyzed generated product using the basic subunit primers, and aligned identically to the Mtb nucleotide sequence. However, only 11 out of the 14 samples generated a product with the acidic subunit primers. These 11 sequences aligned identically to the Mta nucleotide sequence. The venom from the three snakes whose DNA did not amplify with the acidic subunit primers were not recognized by antibodies to Mojave toxin. This suggests that snakes with venom lacking Mojave toxin also lack the productive nucleotide sequence for the acidic subunit in their DNA.

Purification of M5, a fibrinolytic proteinase from Crotalus molossus molossus venom that attacks complement.

Crotalus molossus molossus (northern blacktailed rattlesnake) venom contains agents that affect blood coagulation. A fibrin(ogen)olytic proteinase, called M5, was isolated and purified from this venom by ion exchange chromatography in a two-step procedure. M5 consists of a single non-glycosylated polypeptide chain with a molecular weight of 25 kDa and an isoelectric point of 7.6. It hydrolyses the A alpha and B beta chains of fibrinogen and the alpha and beta chains of fibrin. It also exhibits caseinolytic activity, but has no effect on synthetic substrates cleaved by thrombin, plasmin, kallikrein, or trypsin. The proteolytic activity of the enzyme against fibrinogen, fibrin, and casein is inhibited by ethylenediaminetetraacetic acid (EDTA) and the loss of activity by EDTA treatment can be prevented by addition of Zn2+. This suggests that M5 is a zinc metalloproteinase. M5, at doses of 50 micrograms and higher, induces significant hemorrhage when injected subcutaneously into mice. In addition, it inactivates guinea-pig complement in a dose-dependent fashion and hydrolyses human C2, C3, and C4.

Differences in fibrinolysis and complement inactivation by venom from different northern blacktailed rattlesnakes (Crotalus molossus molossus).

Venom from 72 different Crotalus molossus molossus rattlesnakes was examined for fibrinolysis and for their ability to inactivate human complement. The fibrinolytic activity of the venoms was variable, but smaller (younger) snakes had less fibrinolytic activity than larger (older) snakes. Major differences between the venoms was detected by isoelectric focusing, and reflected in the number and pI of the proteins with fibrinolytic activity. Of the 72 venoms tested, ten had no effect and three had low activity on complement. The rest of the venoms strongly inactivated complement. The snakes with no activity on complement measured 55 cm or less in length, except for one snake which measured 53 cm and completely inactivated complement. Two larger snakes (76 and 84 cm) had a reduced complement-inactivating activity. Some venoms strongly hydrolyzed C2, whereas others had mild or no effect on this complement component. The attack on C3 was variable: some had no effect on C3, while other venoms produced a 125,000 mol. wt protein, which was recognized by antibodies to C3. Only mild hydrolysis of C4 was evident in serum treated with some venoms. No relationship was evident between the venom properties of this species and geographical distribution. Venom variability is an important clinical reality, and is an important consideration when attempting to isolate proteases from this snake species for further study.

Cobra venom cytotoxin free of phospholipase A2 and its effect on model membranes and T leukemia cells.

Membrane-active toxins from snake venom have been used previously to study protein-lipid interactions and to probe the physical and biochemical states of biomembranes. To extend these studies, we have isolated from Naja naja kaowthia (cobra) venom a cytotoxin free of detectable phospholipase A2 (PLA2). The amino acid composition, pI (10.2), and net charge of the cytotoxin compares well with membrane-active toxins isolated from venoms of other cobras. The cytotoxin, shown by a spin label method, associates with PLA2 in buffers at pH values between 7.0 and 5.0, but not at pH 4.0. It is suggested that cytotoxin and PLA2 (pI close to 4.8) associate electrostatically in the native venom. The effect of the cytotoxin on model phospholipid membranes was studied by EPR of spin probes in oriented lipid multilayers and 1H-NMR of sonicated liposomes. The cytotoxin did not significantly affect the packing of lipids in pure phosphatidylcholine (PC) membranes and in PC membranes containing 10 mol% phosphatidic acid (PA) or cardiolipin (CL). However, the cytotoxin induced an increase in membrane permeability and formation of nonbilayer structures in PC membranes containing 40 mol% of PA or CL. The purified cytotoxin was cytocidal to Jurkat cells, but had little effect on normal human lymphocytes. However, both Jurkat cells and normal lymphocytes were killed equivalently when treated with 10(-9) m PLA2 and 10(-5) m cytotoxin in combination. From its effect on model membranes and Jurkat cells, it is suggested that purified cytotoxin preferentially targets and disrupts membranes that are rich in acidic phospholipids on the extracellular side of the plasma membrane.

In vitro evaluation of Pyrularia thionin-anti-CD5 immunotoxin.

The membrane-active peptide, Pyrularia thionin, purified from Pyrularia pubera, was covalently conjugated to an anti-CD5 monoclonal antibody. The membrane-active properties of thionin were not affected by the conjugation. The immunotoxin killed CD5+ lymphocytes in vitro at a concentration of 0.1 nmol/10(7) cells after 2 h of incubation. The immunotoxin also inhibited the proliferation of T cells in vitro, stimulated either by mitogens or in the mixed lymphocyte reaction. It was shown by electron paramagnetic resonance of spin probes and differential scanning calorimetry that the ability of the immunotoxin to perturb the lipid phase of membranes is close to that of unconjugated thionin. The results obtained suggest that Pyrularia-thionin-anti-CD5 conjugate may be useful for graft-versus-host disease therapy and potentially in the treatment of CD5+ leukemia and lymphomas.

Phospholipase A2 and cobra venom cytotoxin Vc5 interactions and membrane structure.

The hydrolytic activity and interaction of acidic and neutral phospholipase A2 (PLA2) with large unilamellar liposomes treated with cobra venom cytotoxin Vc5 (CT Vc5) were studied to more fully understand the modulating effects of cationic membrane-active peptides on PLA2. Studies were done by fluorescence displacement, EPR spin probes, and 31P-NMR. The results showed that CT Vc5 inhibits PLA2 activity on phosphatidylcholine liposomes. Enzymatic activity of both acidic and neutral PLA2's were enhanced on liposomes containing cardiolipin and pretreated with cytotoxin. The cytotoxin, however, inhibited enzyme lipid hydrolysis if these same liposomes were first treated with acidic PLA2. The highest enzymatic activity was found on substrates with nonbilayer lipid packing. Using EPR of spin labeled enzymes, it was shown that CT Vc5 inhibited binding of acidic PLA2 to liposomes and caused displacement of acidic PLA2 from liposomes. No direct interaction was found between CT Vc5 and neutral PLA2.. It is suggested that cytotoxin perturbs packing of lipid molecules in liposomes containing cardiolipin and is responsible for increased catalysis, whereas direct interaction between CT Vc5 and acidic PLA2, inhibits enzyme activity. It is concluded that variability in substrate composition and the chemical nature of both PLA2 and cationic peptide determine whether enzyme activity is affected by substrate packing or by direct enzyme-peptide interaction. Models of interactions of PLA2 with CT Vc5 and phospholipid membranes are presented.

Modulation of phospholipase A2 activity by membrane-active peptides on liposomes of different phospholipid composition.

To determine the influence of variations in both lipid species and lipid packing on phospholipase A2 (PLA2) hydrolytic activity, the activities of two PLA2 isolated from Crotalus molossus molossus venom, were followed on unilamellar liposomes modified by membrane-active peptides. Enzymatic activity was compared with cytolytic activity on human and mouse lymphocytes. Phosphatidylcholine liposomes were hydrolysed better than liposomes containing acidic phospholipids (phosphatidylserine, phosphatidic acid or cardiolipin) or phosphatidylethanolamine. Both membrane-active peptides, cardiotoxin and thionin, inhibited the PLA2 activity on phosphatidylcholine liposomes. The activities of the enzymes were profoundly enhanced on thionin-pretreated liposomes containing phosphatidylserine, and on cardiotoxin-pretreated liposomes containing cardiolipin or phosphatidic acid. Both cardiotoxin and thionin facilitated the cytolytic activities of PLA2 on both human and mouse lymphocytes. Cytolytic activity correlated well with esterase activity. It is proposed that the complex dynamic structure of cell membranes renders a variety of substrate configurations that transiently affect PLA2 activity.

Detection of alkaline phosphatase in venom by blotting methods.

Venom alkaline phosphatase was detected using a blotting method following electrophoresis. The enzyme gave strong reactions in some venoms, but was absent in other venoms, some within the same species. The mol. wt of the enzyme is close to 100,000 and its pI is between 3.6 and 4.8. The enzyme was inactivated by EDTA and 2-mercaptoethanol, and lost activity by freezing and thawing. Endogenous venom alkaline phosphatase can interfere with alkaline phosphatase-based detection methods. Pre-screening for endogenous venom alkaline phosphatase is recommended prior to using alkaline phosphatase-based detection methods when studying snake venom.

Detection of botulinum toxin using an evanescent wave immunosensor.

Using fluorescein isothiocyanate (FITC)-streptavidin, quartz fibre-immobilized antibody (FiAb) and the evanescent wave component of a light beam, detection of Botulinum Toxin-B (BoTX) is described. Exposure of 3-aminopropyltriethoxysilane/glutaraldehyde (APTS/GA) treated quartz fibres to increasing amounts of anti-BoTX Ab indicated toxin binding to increase in a linear fashion up to approximately 125 ng added Ab. Quantitation of bound BoTX and FiAb by Dot-Blot analysis using avidin-Horseradish peroxidase (HRP) conjugation indicated the presence of 0.27 and 0.67 pmoles, respectively. Inclusion of nonbiotinylated BoTX in sampling mixtures reduced fluorescence in a dose-dependent manner over a narrow concentration range (0-300 ng). Exposure of FiAb to a variety of venoms resulted in no reduction of BoTX binding suggesting detection of BoTX via immobilized anti-BoTX Ab to be very specific.

Hemorrhagic and Mojave toxins in the venoms of the offspring of two Mojave rattlesnakes (Crotalus scutulatus scutulatus).

1. The venoms of two Mojave rattlesnakes and those of their offsprings were analyzed for Mojave toxin and hemorrhagic toxin. 2. The venom of one female, collected in Pima County, Arizona, and the venoms of her six offspring contained hemorrhagic toxin but not Mojave toxin (venom B). 3. The venom of the second female, captured in El Paso County, Texas, contained both toxins (A+B venom). Of her 10 offspring, five contained venom with both toxins, two had hemorrhagic toxin only, and three contained neither toxin. 4. Venoms that caused hemorrhage also inactivated complement. A pool of the venoms of the venom B offspring was less toxic than adult pooled venom A.

Detection of sub-nanogram quantities of Mojave toxin via enzyme immunoassay with light addressable potentiometric detector.

Sensitive detection of Mojave toxin (MoTX), a potent neurotoxin isolated from the venom of Crotalus scutulatus scutulatus, was developed using an enzyme immunoassay (EIA) and light addressable potentiometric detection. This EIA utilizes both biotin- and fluorescein-labeled anti-MoTX antibodies to immobilize and detect sub-nanogram to nanogram quantities of toxin. Labeled mono- and polyclonal anti-MoTX antibodies were used alone and/or in combination to determine maximum assay sensitivity. Assays performed using a combination of biotinylated poly- and fluoresceinated monoclonal antibodies produced an assay with a lower detection limit near 2.5 ng. Assays performed using labeled polyclonals alone, or in combination with biotinylated mono- and fluoresceinated polyclonal antibodies, indicated increased sensitivity with a detection limit near 300 pg. In conclusion, we describe an enzyme immunoassay using different labeled antibody schemes which detects sub-nanogram quantities of MoTX via light addressable potentiometric detection.

Variation in the antigenic characteristics of venom from the Mojave rattlesnake (Crotalus scutulatus scutulatus).

Venoms from 31 specimens of the Mojave rattlesnake (Crotalus scutulatus scutulatus) were examined to further characterize reported differences among venoms of this species. Twenty-two venoms were recognized by a monoclonal antibody to Mojave toxin, CSS12. Nine venoms were recognized by CA-P-8, a monoclonal antibody produced against the hemorrhagic venom of C. atrox. Seven of these produced strong hemorrhage in mice and were also recognized by polyclonal antibodies (anti-F5) produced against a fraction of Mojave rattlesnake venom that inactivates serum complement. Fractionated venom revealed that CA-P-8 and anti-F5 recognized different proteins. Two of the venoms recognized by CA-P-8 were not recognized by anti-F5 and produced minimal hemorrhage in mice. This suggests that more than one factor may be necessary to induce strong hemorrhage.

Isolation of a fibrinolytic protease, M4, from venom of Crotalus molossus molossus (northern blacktail rattlesnake).

M4, a fibrinolytic protease, was isolated from the venom of Crotalus molossus molossus. It has a pI of 9.6 and a molecular weight of 27,000. The protease hydrolyzes the A alpha and B beta chains of fibrinogen, and the alpha and beta chains of fibrin. This activity was inhibited by EDTA and restored by Ca2+ or Zn2+, but not Mg2+. The protease hydrolyzed hide power azure and casein, but it had no effect on collagen, hyaluronic acid, complement or synthetic substrates for thrombin, plasmin or kallikrein. Subcutaneous injections into mice with doses as high as 100 micrograms did not cause hemorrhage. This protease may have therapeutic use as a thrombolytic agent.

Isolation of two hemorrhagic toxins from Crotalus basiliscus basiliscus (Mexican west coast rattlesnake) venom and their effect on blood clotting and complement.

1. Two hemorrhagic toxins of mol. wt 27,000 (B1) and 27,500 (B2) and pI 9.8 and 5.2 respectively were isolated from Crotalus basiliscus venom. 2. The two proteinases did not cross-react antigenically. 3. Both toxins caused hemorrhage in mice and each was capable of hydrolyzing hide power azure, casein, collagen and fibrin. 4. B1 hydrolyzed the A alpha, B beta and gamma chains of fibrinogen. B2 hydrolyzed the A alpha and B beta chains of fibrinogen, but not the gamma chain. 5. Both proteinases inactivated guinea pig complement.

Isolation of a hemorrhagic toxin from Mojave rattlesnake (Crotalus scutulatus scutulatus) venom.

A hemorrhagic toxin was isolated from Mojave rattlesnake venom. The isoelectric point of the toxin was 4.7 and its mol. wt was 27,000. Concentrations as low as 2 micrograms injected s.c. in mice caused hemorrhage greater than 5 mm in diameter. The toxin was fibrinogenolytic and hydrolyzed hide powder azure, casein and collagen. The toxin also partially inactivated complement. It had no activity against elastin, fibrin, and the chromogenic substrates S-2805, S-2302 and S-2238. Its esterolytic activity was 3% of the activity of the unfractionated venom. The enzymatic and hemorrhagic activities were inhibited by EDTA. The hemorrhagic toxin was absent or in low quantities in Mojave rattlesnake venoms containing Mojave toxin. Chromatography by HPLC easily distinguishes Mojave rattlesnake venoms into two types by the presence or absence of the hemorrhagic toxin.

Suppression of phytohemagglutinin induced splenocyte proliferation during concurrent infection with Eimeria nieschulzi and Nippostrongylus brasiliensis.

Results suggest that infection with Eimeria nieschulzi (Protozoa) interferes with splenocyte proliferation induced by infection with Nippostrongylus brasiliensis (Nematoda).

Inhibition of calcium channel dihydropyridine receptor binding by purified Mojave toxin.

Mojave toxin, the principal toxic component of the venom of the Mojave rattlesnake Crotalus scutulatus scutulatus, is a protein complex of about 22,000 mol. wt. The mechanism of action of this potent (LD50 = 0.039 micrograms/g, mouse, IV) neurotoxin is a matter of conjecture, but physiologic data suggest a presynaptic site of action with disruption of stimulus-secretion coupling and neurotransmitter release. The selectivity of Mojave toxin's effect on several ion channels involved in neurotransmission was assessed in the present study using competitive radioisotopic binding procedures. Synaptic membranes from rat brain were used to assess the toxin's interaction with Ca++ and Cl- channels while membrane fragments from the Torpedo fish electric organ were used to determine toxin interaction with the nicotinic acetylcholine receptor-coupled Na+ channel. Mojave toxin was found to irreversibly inhibit 3H-nitrendipine binding to dihydropyridine receptors associated with Ca++ channels in rat brain, but had no effect on radioligand binding in the Na+ and Cl- channel assays. Saturation analysis of the binding further showed that the effects of MoTX on dihydropyridine binding were noncompetitive, with MoTX producing a decrease in both the affinity and density of 3H-nitrendipine sites. These results are consistent with the hypothesis that MoTX acts selectively on Ca++ channel function and that this interaction occurs via an allosteric mechanism in which MoTX binds to a membrane site that is topologically distinct from the dihydropyridine receptor.

Analysis of the individual allergens of Russian thistle pollen by an enzyme-linked immunoblotting technique.

Russian thistle pollen extract was analyzed by immunoblots of isoelectric focused and SDS-PAGE gels. Twenty distinct protein bands were recognized by human IgE- and IgG-specific antibodies in the immunoblot from the SDS-PAGE gel. Molecular weights of these allergens ranged from 12.2 kD to 85 kD. Seventeen bands were detected on isoelectric focusing immunoblots with pI from 3.95 to 7.70. Allergic subjects had differing individual patterns of protein band recognition. Immunoblot techniques provide detailed evaluations of the response of allergy subjects to components of crude natural allergens.

The effects of purified Mojave toxin on rat synaptic membrane (Ca+2 + Mg+2)-ATPase and the dihydropyridine receptor.

Effects of purified Mojave toxin on rat synaptic membrane (Ca+2 + Mg+2)-ATPase and dihydropyridine receptor were determined. The toxin was observed to stimulate specifically (Ca+2 + Mg+2)-ATPase approximately two-fold with no effect on Mg+2 dependent ATPase activity. Examination of the effects of increasing amounts of purified Mojave toxin on binding of the calcium channel blocker, nitrendipine, indicated that the addition of 10 micrograms (4.5 X 10(-10) moles) of toxin resulted in greater than 90% inhibition of nitrendipine binding. Furthermore, binding studies revealed the toxin to have little affinity for the ligand indicating its interaction with calcium channel components. Since Mojave toxin has associated with it a phospholipase A2 activity, we investigated the effects of 4-bromophenacylbromide, a known inhibitor of phospholipase A2 activity in order to discern the possible effects of the purified toxin on synaptic membranes. At concentrations previously shown to be inhibitory of purified phospholipase A2 from cobra venom, both ATPase activity and nitrendipine binding of synaptic membranes were significantly inhibited. Thus we cannot rule out the possibility that the endogenous phospholipase activity of the purified toxin is responsible for its effects on the rat brain synaptic functions studied here. Binding studies conducted in the presence of verapamil and diltiazem indicated that the toxin interacts with allosteric sites responsible for regulation of the binding of nitrendipine. Although we have not tested the effects of Mojave toxin on other ion channels and/or receptors, results presented here suggest the potential usefulness of this toxin as a molecular probe of the calcium channel complex.

Monoclonal antibodies to Mojave toxin and use for isolation of cross-reacting proteins in Crotalus venoms.

Hybridomas secreting monoclonal antibodies against Mojave toxin were established. The antibodies were used for identifying cross-reacting proteins in individual C. s. scutulatus and other Crotalus venoms and to isolate Mojave toxin. The antibodies recognized five bands with a pI range from 5.1 to 6.1 in immunoblots of electrofocused crude venom and Mojave toxin purified by immunoaffinity chromatography. The specificity of the antibodies was for the basic subunit of the toxin, which resolved into four bands of pI between 9.3 and 9.6. Individual C. s. scutulatus venoms of snakes from Texas and southern Arizona had multiple bands with pI's ranging from 4.9 to 6.3. Cross-reacting proteins were also recognized by the antibodies in the electrophoresed venoms of C. basiliscus, C. d. durissus, C. d. terrificus, C. h. horridus and C. v. concolor, and may be isolated by immunoaffinity chromatography with the monoclonal antibodies.