Studies of an acidic cardiotoxin isolated from the venom of Mojave rattlesnake (Crotalus scutulatus).

A major lethal protein was isolated from the venom of Mojave rattlesnake (Crotalus scutulatus) by successive purification in DEAE column chromatography and isoelectric focusing. This homogeneous and monomeric form of toxin is designated as "Mojave toxin". Unlike basic neurotoxins or cytotoxins isolated from venoms of cobras, kraits and sea snakes, the Mojave toxin is an acidic protein with an isoelectric point of 4.7. The toxin is also different from crotoxin (from Crotalus durissus terrificus) which consists of both acidic and basic components. The molecular weight determined by Sephadex G-75 column chromatography resulted in a value of about 22 000. A singel protein band with a molecular weight of about 12 000, was observed after sodium dodecyl sulfate gel electrophoresis of the reduced Mojave toxin. Isoelectric focusing gel in the presence of 8 M urea also showed a single protein band, suggesting that the toxin is composed of subunits. Unlike the neurotoxic nature of the basic proteins from the venoms of Elapidae and sea snakes (Hydrophiidae) and crotoxin, Mojave toxin is cardiotoxic rather than neurotoxic. It is very likely that venoms of all rattlesnakes from North and Central America contain Mojave toxin as the common toxin.

Assignment of the aromatic 1H-NMR resonances of myotoxin a isolated from the venom of Crotalus viridis viridis.

The 400 MHz 1H-NMR spectrum of myotoxin a from the venom of Crotalus viridis viridis is described. The identification of spin systems in the aromatic region corresponding to the six aromatic residues of myotoxin a was completed using both one- and two-dimensional NMR spectroscopy and the pH dependence of chemical shifts. Assignments of these spin systems to specific residues was possible for the singly occurring amino acids Tyr-1 and Phe-12. Resonances from Tyr-1, His-5 and His-10 were shifted significantly from their random coil values in a pH-dependent manner. These shift perturbations were deemed evidence of a helical arrangement of the amino terminal region which placed these residues in close proximity to each other.

The complete sequence of the acidic subunit from Mojave toxin determined by Edman degradation and mass spectrometry.

Mojave toxin, a heterodimeric, neurotoxic phospholipase complex from Crotalus scutulatus scutulatus, is one of a group of closely related rattlesnake toxins for which much structural information is still lacking. The complete amino-acid sequence of the acidic subunit from Mojave toxin was determined. The three individual peptide chains, derived from the acidic subunit by reductive alkylation, were separated by high-performance liquid chromatography. Fragmentations of the A and B chains were done using specific proteinases and the resulting peptide mixtures were fractionated by reverse-phase high-performance liquid chromatography. Sequence analyses on the intact chains and the fragments from digests were done by automated Edman degradation, carboxypeptidase Y degradation and triple-quadrupole and tandem-quadrupole Fourier-transform mass spectrometry. The sequence for each acidic subunit chain is very similar to the corresponding chain from the related neurotoxin complex, crotoxin, and overall the sequence is similar to the sequences of group I and II phospholipases A2. The N-terminus of the B chain is blocked by pyroglutamic acid. The existence of two distinct and closely related C chains was established. It is unlikely that the small sequence difference can account for the isoforms that are present in purified Mojave toxin and in unfractionated venom.

The effects of myotoxin from midget faded rattlesnake (Crotalus viridis concolor) venom on neonatal rat myotubes in cell culture.

Neonatal rat myoblasts were isolated and grown in culture until they fused into multinucleated myotubes. A small percentage of the myotubes showed spontaneous contractions when maintained in Dulbecco's Modified Eagle's Medium with 10% fetal bovine serum. Incubation of mature myotubes (at least 3 days after fusion) with myotoxin II from Crotalus viridis concolor venom at a concentration as low as 18.5 nM caused a marked increase in the number of myotubes demonstrating contractile activity. The increase was apparent within 24 hr of myotoxin application. The response of the myotubes appeared to be specific since, of the proteins tested, only native myotoxins caused the increase in contractile activity. This tissue culture system offers a rapid screening assay that requires less time and fewer animals than the assays currently in use for determining myotoxic activity.

Mojave toxin affects fusion of myoblasts and viability of myotubes in cell cultures.

Mojave toxin is a neurotoxic, heterodimeric phospholipase isolated from the venom of Crotalus scutulatus scutulatus. Responses of primary rat muscle cell cultures and clonal muscle cell lines to treatment with Mojave toxin and its constituent subunits were examined. Continuous exposure of cells to 0.5 microM or 1.0 microM Mojave toxin or the basic subunit, added 24 hr after plating, prevented fusion of primary myoblasts and C2 myoblasts to multinucleate myotubes. Under the same experimental conditions, some myotube formation was observed when RMo cells were used, but the number and size of the myotubes were reduced substantially compared to untreated controls. The addition of Mojave toxin to established myotubes that arose from differentiation of primary myoblasts or C2 myoblasts essentially led to total disappearance of the myotubes from the cell layer within 48 hr. Myotubes from RMo cells treated in the same manner, however, did not disappear, but they were smaller and less numerous than comparable controls. Similar results were generated by exposure of myotubes to the basic subunit of Mojave toxin under the same conditions. The underlying layer of mononucleate cells was retained in both instances. Toxin-free cultures continued to develop in the usual manner. Treatment with 1.0 microM concentrations of the acidic subunit, pancreatic phospholipase A2 or a non-neurotoxic phospholipase from Naja naja atra gave results indistinguishable from untreated control cultures.

Mojave toxin: rapid purification, heterogeneity and resistance to denaturation by urea.

This report establishes that purified Mojave toxin prepared from the snake venom of Crotalus scutulatus scutulatus contains multiple heterogeneous dimers (isoforms) differing slightly in isoelectric points. This conclusion is based upon chromatographic, immunological, sodium dodecyl sulfate--polyacrylamide gel electrophoretic and polyacrylamide isoelectric focusing experiments. The Mojave toxin-related proteins were rapidly purified from venom via a single chromatography step. Generation of Mojave toxin-related proteins from isolated subunits and immunoblots of these proteins subsequent to electrophoretic separation demonstrate that each of the proteins consists of acidic and phospholipase basic subunits. The analysis of venom in narrow range polyacrylamide isoelectric focusing gels at varying concentrations of urea, in conjunction with immunoblots utilizing antibodies specific to the basic subunit, demonstrates that the isoforms of Mojave toxin are native and not artifacts from isolation procedures. Analyses of venoms from Crotalus scutulatus scutulatus individuals indicate that each snake produces multiple isoforms of the neurotoxin. Additionally, the same predominant isoform of Mojave toxin is present in both individual and commercial venoms. The heterogeneity of the Mojave toxin-related proteins is largely due to differences in the acidic subunits and some of the forms may reflect post-translational processing of the protein. The Mojave toxin-related proteins demonstrate a resistance to urea denaturation by characteristically entering and focusing in polyacrylamide isoelectric focusing gels containing 0-6 M urea, but dissociating to constituent subunits in 8 M urea. Experimental evidence suggest that salt bridges may be important in stabilization of the Mojave toxin complex.

Characterization of the two myotoxin a isomers from the prairie rattlesnake (Crotalus viridis viridis) by capillary zone electrophoresis and fluorescence quenching studies.

The two myotoxin a isomers from the venom of the prairie rattlesnake Crotalus viridis viridis have different isoelectric points, as determined by capillary zone electrophoresis. The pI values are 10.50 and 10.57, respectively, and both are higher than the previously reported pI value for myotoxin a. The difference in the isoelectric points between the two isomers is attributed to altered surface charge as a result of the conformational change in myotoxin a. Both isomers exist in crude venom, discounting the possibility that they are artifacts formed during the purification process. Fluorescence quenching of myotoxin a reveals heterogeneity of the tryptophans, possibly due to different environments. The fraction of the total tryptophan fluorescence quenched by iodide is 81% and is attributed to solvent-accessible tryptophan residues at the protein surface. The 19% non-quenchable tryptophans probably represent residues that are shielded from the solvent exposure. The ratio of buried to exposed tryptophans is similar to the ratio of isomers seen by capillary zone electrophoresis and reverse-phase high-performance liquid chromatography (c. 1 : 4).

Characterization of two arginine ester hydrolases from Mojave rattlesnake (Crotalus scutulatus scutulatus) venom.

Two arginine ester hydrolases, designated AAEI and AAEII, from the venom of Crotalus scutulatus scutulatus have been investigated. The amino acid content of both enzymes were very similar and both esterases contained carbohydrate. Following treatment of AAEI and AAEII with neuraminidase, both enzymes migrated identically in two electrophoresis systems and one electrofocusing system. The esterase activities of both enzymes were optimally active in the range pH 8.0-8.5. Neither esterase hydrolyzed casein, hemoglobin (Hb) or alpha-N-benzoyl-DL-arginine-p-nitroaniline (BAPNA), yet both AAEI and AAEII hydrolyzed alpha-N-benzoyl-L-arginine ethyl ester (BAEE), alpha-N-benzoyl-L-arginine methyl ester (BAME), p-tosyl-L-arginine methyl ester (TAME) and acetylphenylalanylarginine methyl ester (Ac-Phe-Arg-OMe). The esterase activities of the two enzymes were inhibited by serine specific reagents and benzamide, but not by EDTA or soybean trypsin inhibitor. The Km values for each enzyme with alpha-N-benzoyl-L-arginine ethyl ester and acetylphenylalanylarginine methyl ester were determined. Neither esterase displayed thrombin-like or fibrinolytic activities. Both AAEI and AEII possessed kinin releasing activity as shown by the twitch response of an isolated rat uterus. The N-terminal sequences of AAEI and AAEII were identical and both enzymes sequences were similar to other arginine esterases from crotalid venoms. The properties of AAEI and AAEII are compared to several other arginine esterases possessing kallikrein-like activities which have been isolated from snake venoms.

Antigenic relationships between Mojave toxin subunits, Mojave toxin and some crotalid venoms.

Immunochemical responses of a number of pit viper venoms to antibodies derived separately from the acidic and basic subunits were investigated by enzyme linked immunosorbent assay (ELISA) and Ouchterlony immunodiffusion. The polyclonal antisera to the basic subunit were generated in rabbits, whereas mouse hybridoma cell cultures were used to produce antibodies to the acidic subunit. The immunochemical response of a venom correlated well with published values for LD50 dose for the test venom. Many venoms that elicited a positive response with antiserum to the basic subunit also reacted strongly with the hybridoma derived antibodies to the acidic subunit. The data support the conclusion that crotalid venoms which are more lethal have in common a potent venom component that is immunochemically related to Mojave toxin.

Effects of myotoxin alpha on fusion and contractile activity in myoblast-myotube cell cultures.

Cultured myoblasts and moytubes were used to study the effects of purified myotoxins from rattlesnake venoms. Standard cell culture techniques were used to establish and maintain primary cultures derived from neonatal rat tissue and two clonal cell lines, rat RMo cells and mouse C2 cells. Toxin concentrations, ranging from 0.04 to 1.0 microM, were added to the cultures at various times under distinct, well-defined conditions. Addition of myotoxin alpha to primary myoblast cultures did not appear to affect the fusion process, whereas similar experiments with two clonal cell lines produced larger myotubes when contrasted with untreated control cultures, particularly with RMo cells. The myotubes derived from primary cell cultures twitched spontaneously but the twitching ceased when the medium was replaced with a serum-free chemically defined incubation medium. Addition of myotoxin alpha to the primary myotubes incubated with serum-free defined medium caused the myotubes to twitch again. Derivatives of myotoxin alpha were prepared by reactions with tetranitromethane and with iodoacetic acid, the latter under reducing and non-reducing conditions. The resulting products, purified but not chemically characterized, were nearly devoid of activity when primary cultures were used to test activity.

Amino acid sequences of myotoxins from Crotalus viridis concolor venom.

Myotoxins I and II were isolated from the venom of Crotalus viridis concolor. Complete sequences were derived for each reduced, alkylated toxin with data obtained by a single run on a gas phase sequencer and from fragments derived by cyanogen bromide cleavage. The results demonstrate that microheterogeneity is present in myotoxin II. The newly established sequences were compared with 3447 protein sequences in the Protein Information Resource database. The only homologous proteins found were other known myotoxins from rattlesnake venoms, namely myotoxin a, crotamine and peptide C.

Mojave rattlesnake (Crotalus scutulatus scutulatus) venom: enzyme activities and purification of arginine ester hydrolases.

Venom from the Mojave rattlesnake was fractionated on DEAE Sephadex into 12 fractions (MD-1-12). Each fraction was assayed for five enzymatic activities, all known to occur in crotalid venoms. Phosphomonoesterase activity was not present in either the crude venom or any of the fractions. L-Amino acid oxidase activity was found in several fractions (MD-4-9), being eluted from the column as a broad peak of activity. Phosphodiesterase activity was found in MD-1-2, eluting from the column as a single peak of activity. Phospholipase activity was fractionated into three peaks of activity in MD-2-4, MD-7 and MD-9. The phospholipase activity in MD-9 was associated with Mojave toxin. Arginine ester hydrolase activity was distributed as several peaks of activity throughout MD-1-9. Two arginine ester hydrolases (AAEI, AAEII) were isolated from Mojave venom by fractionation on DEAE Sephadex followed by chromatofocusing chromatography and affinity chromatography. They were purified to specific activities of 60 U/mg (AAEI) and 36.1 U/mg (AAEII) with BAEE as substrate at pH 7.5. This procedure showed per cent yields of 5.0% for AAEI and 1.0% for AAEII. The two proteins were homogeneous by PAGE, narrow range isoelectric focusing and SDS gel electrophoresis. Both proteins were acidic, with pI values equal to 4.7 (AAEI) and 4.4 (AAEII). The molecular weights as determined by SDS gel electrophoresis were 33,200 for AAEI and 34,700 for AAEII.

Evidence for isomerization in myotoxin a from the prairie rattlesnake (Crotalus viridis viridis).

Myotoxin a, from the venom of the prairie rattlesnake, Crotalus viridis viridis, exists as a temperature-dependent equilibrium of two interconverting forms. Reverse-phase high-performance liquid chromatography (RP-HPLC) shows that the two forms interconvert slowly enough at 25 degrees C to be seen as two separate peaks with a molar ratio of c. 1:4. Each peak can be isolated and individually injected to give the same two peaks in the same ratio of areas. The two peaks merge during chromatography at elevated temperatures, indicating an increase in the rate of interconversion. At low temperature, c. 5 degrees C, the individual peaks can be isolated and maintained for several days without reaching equilibrium. Mass analysis by matrix-assisted laser desorption ionization (MALDI) time-of-flight mass spectrometry shows that myotoxin a is present in both RP-HPLC peaks, suggesting that the two resolved forms are conformational isomers. Capillary zone electrophoresis (CZE) also shows two resolved, but interconvertible peaks over a range of pH values. Furthermore, RP-HPLC chromatograms of myotoxin a at concentrations from 0.013 mM to 0.41 mM maintain a consistent ratio of peak areas, without evidence of dimerization. Two-dimensional 1H-NMR nuclear Overhauser enhancement spectroscopy indicates the presence of a cis-proline peptide bond, consistent with an equilibrium mixture of cis-trans isomers; however, addition of peptidyl-prolyl cis-trans isomerase (PPI) does not enhance the rate of equilibration of the RP-HPLC peaks isolated at c. 5 degrees C.

Purification of rabbit liver guanine aminohydrolase.

Rabbit liver guanine aminohydrolase has been purified 1250-fold by utilization of an affinity chromatographic separation on 9-(p-aminoethoxyphenyl) guanine-Sepharose with 50% recovery of activity. Polyacrylamide gel electrophoresis of the purified preparations revealed several protein bans which corresponded to regions of enzyme activity measured on gels which had been run under the same conditons. Gel concentration studies of the protein migration rate showed that the protein bans differed in molecular size. The minimum molecular weight was 100,000 from gel permeation chromatography studies. The pH optimum was near pH 8 and the Km, with guanine as substrate was 5.6 x 10-6 M. The latter values are in close agreement with partially purified preparations described in the literature.

Studies of rabbit brain, intestine and liver guanine aminohydrolase.

Guanine aminohydrolase (EC 3.5.4.3) from rabbit brain, intestine, and liver has been purified to homogeneity by affinity chromatography at room temperature and 0-4 degrees C. In all cases the recovery and fold purification was greatest for purification at room temperature. Each enzyme has a subunit mol. wt of 49,500 and crosslinking studies revealed the native form to be a dimer. The amino acid analysis showed a high content of asx, glx, ser, gly and leu; the pI was 5.0 for each prep. The Michaelis constants at pH 8.0 were 6.1, 6.1 and 5.8 X 10(-6) M for brain, intestine and liver GAH respectively. The enzymes had a broad pH activity profile with an optimum of pH 8.0-8.5.

The primary structure of ammodytin L, a myotoxic phospholipase A2 homologue from Vipera ammodytes venom.

A new myotoxic phospholipase A2 homologue, having a serine residue in position 49 instead of highly conserved aspartic acid, was found in the venom of Vipera ammodytes. The primary structure revealed additional mutations in the positions important for enzymatic activity. Tyr28 is exchanged for a histidine and Gly33 for asparagine. These changes render earlier-reported weak enzymatic activity unlikely. The role of this rather abundant venom fraction is apparently in myotoxicity, which was confirmed in the muscle-cell culture from neonatal rats. The muscle-cell culture proved to be a good tool to investigate the effects of various myotoxins on muscle cells.

Purification and chemical characterization of the major neurotoxin from the venom of Pelamis plautrus.

A major toxin was isolated from the venom of the sea snake Pelamis platurus (yellow-bellied sea snake) by Sephadex G-50 and carboxymethylcellulose column chromatography. The LD50 of the pure toxin (Pelamis toxin a) was 0.044 mug/g in mice representing a tenfold increase in toxicity after purification. The toxin was homogeneous in acrylamide disc gel electrophoresis and eluted as a single peak after isoelectric focusing in a sucrose density gradient column. The isoelectric point was 9.69; thus it is a highly basic protein. The toxin contained 55 amino acid residues with four disulfide linkages. When all disulfide linkages were reduced and alkylated, the toxic action of the pure toxin disappeared leading to the conclusion that the disulfide bonds of the neurotoxin were essential for toxic action.