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I I Kaiser

Publications and source records attributed to I I Kaiser.

At least 19 recordsLinked to original sources
PubMed1997

Toxins isolated from the venom of the Brazilian coral snake (Micrurus frontalis frontalis) include hemorrhagic type phospholipases A2 and postsynaptic neurotoxins.

Toxins isolated from the venom of the Brazilian coral snake (Micrurus frontalis frontalis) include hemorrhagic type phospholipases A2 and postsynaptic neurotoxins. Toxicon 35, 1193-1203, 1997.-Two sets of proteins have been purified from the venom of the Brazilian coral snake, Micrurus frontalis frontalis. One set has mol. wts, as shown by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), in the 8000-13,000 range and includes some proteins which are toxic to mice and others which are not. These proteins appear to be isoforms of postsynaptic toxins. The other set shows phospholipase A2 (PLA2) activity and the toxic members of this set promote hemorrhage in mice in a manner closely resembling that produced by PLA2s isolated from the venom of the Australian tiger snake (Notechis scutatus scutatus). These PLA2s migrate on SDS-PAGE with apparent mol. wts in the 18,000-22,000 range which is characteristic of PLA2s that have an alpha-helix D similar to pancreatic PLA2s. Elapid venom PLA2s of the type which typically migrate on SDS-PAGE with mol. wts in the 13,000-16,000 range and do not have alpha-helix D have not been detected in M. f. frontalis venom.

Animals↗
PubMed1996

Influence of ionizing radiation on crotoxin: biochemical and immunological aspects.

Irradiation of crotoxin and its subunits with 2000 Gy of gamma-rays from 60Co source leads to aggregation and generation of lower mol. wt breakdown products. Aggregates separated by gel filtration retain at least part of their higher-ordered structure, based on their reactivity with monoclonal antibodies known to react with conformational epitopes in native crotoxin. These same aggregates can serve as antigens to raise antisera that cross-react and neutralize crotoxin. Compared with native crotoxin, aggregates appear less myotoxic, are largely devoid of phospholipase activity, and are virtually non-toxic in mice. These results indicate that irradiation of toxic proteins can promote significant detoxification, but still retain many of the original antigenic and immunological properties of native crotoxin.

Animals↗
PubMed1996

A phospholipase A2-like pseudogene retaining the highly conserved introns of Mojave toxin and other snake venom group II PLA2s, but having different exons.

Mojave toxin is a neurotoxic, heterodimeric phospholipase A2 (PLA2) from the venom of the Mojave rattlesnake (Crotalus scutulatus scutulatus) and is characteristic of all rattlesnake presynaptic neurotoxins. Here, we describe a phospholipase A2 pseudogene (psi-Mtx) located 2,000 nucleotides upstream, and on the opposite DNA strand, from a gene for Mojave toxin acidic subunit (Mtx-a). The pseudogene lacks the first exon and a few segments of noncoding DNA found in functional snake venom PLA2 genes, but does have the coding information for a complete PLA2 protein. psi-Mtx retains the unusual gene sequence similarity pattern found in functional viperid PLA2 genes. When compared to genes from C. s. scutulatus and the Hahn snake (Trimeresurus flavoviridus), psi-Mtx shows strong conservation of nocoding regions and variable protein-coding regions. Although the nocoding regions of psi-Mtx are conserved with respect to other viperid PLA2 genes, the three exons code for a unique PLA2-like protein similar in sequence to ammodytoxin b found in the venom of the western sand viper (Vipera ammodytes ammodytes). The structure of these genes suggests a common ancestor for all viperid PLA2 genes. Phylogenetic analysis of psi-Mtx, Mtx-a, Mtx-b, pgPLA 1a, and pgPLA 1b suggest that psi-Mtx diverged from an ancestral sequence before the presumed gene duplication event leading to Mtx-a and Mtx-b. However, analysis of the basis of coding regions alone gives a conflicting result.

Amino Acid Sequence↗
PubMed1995

Specificity and binding affinity of an anti-crotoxin combinatorial antibody selected from a phage-displayed library.

A crotoxin-specific, monoclonal, high-affinity, single-chain antibody variable region (scFv) was generated by combinatorial methods using Pharmacia's Recombinant Phage Antibody System. A high-affinity clone, designated A10G, was selected, and its DNA sequence was determined. Protein A10G showed high reaction specificity, with only the closely related rattlesnake neurotoxins, concolor toxin and Mojave toxin, showing cross-reactivity out of eleven group II phospholipase A2s (PLA2s) screened. No group I PLA2s cross-reacted in enzyme-linked immunosorbent assays. The gene coding for A10G was subcloned into an expression vector, and the resulting expressed nonfusion protein, designated A10GPE, was renatured and purified to apparent homogeneity. Dissociation constants of A10G with intact crotoxin and crotoxin basic subunit were determined to be 7 x 10(-10) and 6.8 x 10(-9) M, respectively. When A10GPE was preincubated with either the basic subunit or intact crotoxin at molar ratios of up to 5:1, no inhibition of phospholipase activity was observed. Expressed protein, however, could partially neutralize the lethality of Mojave toxin, a crotoxin homolog, in mice.

Amino Acid Sequence↗
PubMed1995

Amino acid sequence of a new type of toxic phospholipase A2 from the venom of the Australian tiger snake (Notechis scutatus scutatus).

Venom from the common tiger snake, Notechis scutatus scutatus, contains several toxic acidic proteins which promote hypotension and hemorrhage in mice. One of these toxins, HTe, has a phospholipase A2 (PLA2) amino acid sequence. It contains 125 amino acids rather than the 119/120 found in other N. s. scutatus PLA2s, because it also has the loop of residues (62-66) found in helix D of pancreatic PLA2s, the gamma-subunit of taipoxin, and the D-subunit of textilotoxin. High sequence identity is found between the first 57 and the last 25 amino acids of HTe and other N. s. scutatus PLA2s. In the central section containing the pancreatic loop and the beta-wing, sequence similarity with other N. s. scutatus PLA2s is low. The beta-wing amino acids are highly homologous to taipoxin-gamma. HTg, an isoform of HTe, has a sequence almost identical to that of HTe in the central section. Neuropharmacological and neurophysiological studies show that HTe blocks neuromuscular transmission, but it does not produce blockade by virtue of a selective action on nerve endings. Instead, the toxin acts both on nerve and on muscle. Unlike taipoxin-gamma and textilotoxin-D, HTe and HTg are not glycosylated and are not otherwise modified. HTe, HTg, and the other acidic proteins hydrolyze the synthetic PLA2 substrate, 3-octanoyloxy-4-nitrobenzoic acid, as well as L-alpha-phosphatidylcholine.

Amino Acid Sequence↗
PubMed1995

Anions and the anomalous gel filtration behavior of notexin and scutoxin.

Based on their mol. wts, notexin and scutoxin elute later than expected from gel filtration columns in multiple peaks [Francis et al. (1991) Toxicon 29, 85-96]. Notexins present in these peaks have identical amino acid sequences and unmodified amino acid side-chains. Scutoxin is an isoform of notexin which contains arginine at position 16 and glutamate at position 82. Like notexin, it also elutes in different fractions on a gel filtration column, yet the fractions show identical amino acid sequence. This perplexing chromatographic behavior appears to be caused by the association of these proteins with different anions, since dissolving notexin in buffers containing different anions produces up to a 30% change in elution volume. Certain anions promote an apparent reduction in the interaction of notexin with gel filtration matrix, hence earlier elution. These anions include citrate, 3-phosphoglycerate and 2-phosphoglycerate, which also inhibit the PLA2 activity of notexin. However, even under conditions which minimize protein-matrix interaction the toxins elute later than expected based on their mol. wt.

Amino Acid Sequence↗
PubMed1995

Amino acid sequences of a heterodimeric neurotoxin from the venom of the false horned viper (Pseudocerastes fieldi).

The main toxic component of the venom of the false horned viper, Pseudocerastes fieldi, is a heterodimeric neurotoxin composed of a basic subunit, Cb II, and one of two acidic subunits, either Cb I alpha or Cb I beta. The nontoxic acidic subunit increases the toxicity of the basic subunit. Both subunits have phospholipase A2 (PLA2) amino acid sequences. Cb I alpha and Cb I beta themselves are inactive towards phosphatidylcholine and when complexed with Cb II promote a delay in the onset of phospholipase activity of Cb II. Cb I alpha and Cb I beta do hydrolyze the synthetic substrate, 3-octanoyloxy-4-nitrobenzoic acid, but at < 1% the rate of Cb II. Comparisons of the amino acid sequences of Cb II and Cb I alpha with the corresponding acidic and basic subunits of other heterodimeric neurotoxins show high amino acid sequence identity. Some of the amino acids which are different between the acidic and basic subunits are in highly conserved sequences in their respective types of PLA2. This suggests that these amino acid changes in the conserved regions are important for the structure and function of the heterodimeric proteins.

Amino Acid Sequence↗
PubMed1994

Genomic sequences encoding the acidic and basic subunits of Mojave toxin: unusually high sequence identity of non-coding regions.

Mojave toxin (Mtx) is a heterodimeric, neurotoxic phospholipase A2 (PLA2) found in the venom of the Mojave rattlesnake, Crotalus scutulatus scutulatus, and is characteristic of all rattlesnake presynaptic neurotoxins. This paper describes the isolation and nucleotide (nt) sequence of the genomic clones encoding both the non-neurotoxic, non-enzymatic acidic subunit (Mtx-a) and the toxic, PLA2-active basic subunit (Mtx-b), and compares their structures. Both cloned genes shared virtually identical overall organization, with four exons separated by three introns, which were inserted in the same relative positions of the genes' coding regions. The exon/intron structure was similar to that reported for mammalian PLA2 genes. Most remarkable was the high degree of nt sequence identity between Mtx-a and Mtx-b. While the exons shared about 70% identity, the introns were greater than 90% identical and the 5' and 3' untranslated and flanking regions were greater than 95% identical. These findings support our earlier suggestion [Aird et al., Biochemistry 24 (1985) 7054-7058] that the genes coding for the two subunits arose from a common ancestor. There has clearly been a strong selection on the nt sequence of the non-coding regions during this evolutionary process. This is the first report of genomic sequences of PLA2-like proteins from snakes.

Amino Acid Sequence↗
PubMed1994

Primary structures of two proteins from the venom of the Mexican red knee tarantula (Brachypelma smithii).

Venom of the Mexican red knee tarantula (Brachypelma smithii) was fractionated by gel filtration over Sephadex G-50 Fine. Small polypeptides present in the second and third peaks were subfractionated by cation exchange and reversed-phase FPLC. One major, basic protein was isolated and sequenced from each G-50 fraction using a gas-phase protein sequencer. Primary structures were completed and confirmed using tandem mass spectrometry and carboxypeptidase digestions. Protein 1 contains 39 residues, including six cysteine residues in three disulfide bonds. It is identical to one of the isoforms of ESTX from the venom of the tarantula Eurypelma californicum. Brachypelma smithii Protein 5 contains 34 residues, including six cysteine residues in three disulfide bonds. Disulfide bond assignments for both proteins are provided. Protein 5 shows most similarity with toxin Tx2-9 from the Brazilian 'armed' spider, but only displays 41% sequence identity. Similarities with other proteins are lower. Proteins 1 and 5 appear unrelated to each other.

Alkylation↗
PubMed1993

Proteins isolated from the venom of the common tiger snake (Notechis scutatus scutatus) promote hypotension and hemorrhage.

Notechis scutatus scutatus venom contains several toxic acidic proteins called HTa-i which promote hypotension and hemorrhage in mice. They have apparent mol. wts in the 18,000-21,000 range, i.v. LD50 values between 0.5 and 1.5 micrograms/g, and no detectable phospholipase, arginine esterase, proteolytic or hemolytic activities. A polyclonal antibody raised against HTg binds to other purified proteins, suggesting that they are isoforms of the same protein. Many other elapid crude venoms contain proteins which recognize the polyclonal antibody raised against HTg. Crotalid and viperid crude venoms do not recognize this antibody, although some of their component proteins are known to exhibit hypotensive and hemorrhagic activities. A combination of gel-filtration on Sephacryl S-200, cation-exchange and anion-exchange chromatography allows isolation of the N. s. scutatus proteins in high purity. They are the first hypotension-inducing proteins to be purified from an Australian elapid.

Animals↗
PubMed1993

Inhibition of metalloproteinases in Bothrops asper venom by endogenous peptides.

Bothrops asper venom contains a variety of degradative enzymes, including metal-ion dependent proteinases as well as low molecular weight peptides. Two of these peptides, pyroglutamate-glutamine-tryptophan (pEQW) and pyroglutamate-asparagine-tryptophan are present in crude venom at concentrations of about 4.5 and 1 mM, respectively. Proteinase fractions from B. asper are inhibited from digesting oxidized insulin B-chain in vitro by both of these tripeptides with an IC50 for pEQW of approximately 0.3 mM. Digestion of purified myotoxin MIII from B. asper venom is also inhibited in vitro by pEQW, suggesting that similar inhibition of proteinase activities probably occurs in the venom gland. Inhibitory peptides present in venom allow snakes to be protected from their own toxic proteinases and inhibit hydrolysis of venom proteins during storage in the venom gland. Upon dilution, such as when venom is injected into prey, peptide inhibitors dissociate from the proteinase and allow their activation. A simple procedure for isolation of these inhibitory peptides is described.

Amino Acid Sequence↗
PubMed1993

Identification of the site at which phospholipase A2 neurotoxins localize to produce their neuromuscular blocking effects.

Experiments were conducted on mouse hemidiaphragm preparations using five phospholipase A2 neurotoxins of differing chain structures and antigenicities [notexin (one chain); crotoxin (two chains not covalently bound), beta-bungarotoxin (two chains covalently bound); taipoxin (three chains), and textilotoxin (five chains; one copy each of three chains and two copies of a fourth chain)]. Three clostridial neurotoxins (botulinum neurotoxin types A and B, and tetanus toxin) were used in comparison experiments. Phospholipase A2 neurotoxins produced concentration-dependent blockade of neuromuscular transmission. There was no obvious relationship between chain structure and potency, but there was an indication of a relationship between chain structure and binding. The binding of notexin was substantially reversible, the binding of crotoxin was slightly reversible, and the binding of beta-bungarotoxin, taipoxin and textilotoxin was poorly reversible. Experiments with neutralizing antibodies indicated that phospholipase A2 neurotoxins became associated with binding sites on or near the cell surface. This binding did not produce neuromuscular blockade. When exposed to physiological temperatures and nerve stimulation, bound toxin disappeared from accessibility to neutralizing antibody. This finding suggests that there was some form of molecular rearrangement. The two most likely possibilities are: (1) there was a change in the conformation of the toxin molecule, or (2) there was a change in the relationship between the toxin and the membrane. The molecular rearrangement step did not produce neuromuscular blockade. At a later time there was onset of paralysis; the amount of time necessary for onset of blockade was a function of toxin concentration. Phospholipase A2 neurotoxins were not antagonized by drugs that inhibit receptor-mediated endocytosis. In addition, phospholipase A2 neurotoxins did not display the pH-induced conformational changes that are typical of other endocytosed proteins, such as clostridial neurotoxins. However, phospholipase A2 neurotoxins were antagonized by strontium, and this antagonism was expressed against toxins that were free in solution and toxins that were bound to the cell surface. Limited antagonism was expressed after toxins had undergone molecular rearrangement, and no antagonism was expressed after toxin-induced neuromuscular blockade. The cumulative data suggest that phospholipase A2 neurotoxins are not internalized to produce their poisoning effects. These toxins appear to act on the plasma membrane, and this is the site at which they initiate the events that culminate in neuromuscular blockade.

Ammonium Chloride↗
PubMed1992

Citrate is an endogenous inhibitor of snake venom enzymes by metal-ion chelation.

Citrate levels in selected snake venoms were determined by an enzymatic assay coupled to NADP+ reduction. Citrate concentrations in different viper venoms (n = 5) varied from 95 to 150 mM, in crotalids (n = 3) from 63 to 142 mM, and in elapids (n = 4) from 17 to 163 mM. In Bothrops asper venom Ca(2+)-ion concentrations varied from 2.5 to 3.6 mM, suggesting that the high relative citrate levels may serve to chelate endogenous divalent metal cations, thereby inactivating divalent cation requiring enzymes. Control experiments with B. asper phospholipase A2 MIII in the presence of 2.5 mM Ca2+, showed that the enzyme is completely inhibited by 20 mM citrate. Crotalus adamanteus 5'-nucleotidase and phosphodiesterase are also inhibited 100 and 75%, respectively, by 100 mM citrate. By forming complexes with divalent metal ions, citrate markedly reduces the activities of selected enzymes in snake venoms. Secretion of high concentrations of citrate may represent an important mechanism by which snakes protect themselves against the toxic effects of their own venoms.

5'-Nucleotidase↗
PubMed1992

Citrate is a major component of snake venoms.

Citrate has been identified as a major component of snake venoms by gas liquid chromatography and mass spectrometry. The venoms of Bothrops asper, Crotalus atrox, Crotalus viridis viridis, Crotalus adamanteus, Sistrurus miliarius barbouri, Crotalus horridus horridus, Agkistrodon contortrix mokasen, Agkistrodon contortrix contortrix and Agkistrodon piscivorus piscivorus contain citrate at concentration levels which can serve as effective buffers. Calcium, magnesium, zinc, iron, sodium and potassium salts of citrate would be constituents of venom.

Animals↗
PubMed1991

Myotoxin II from Bothrops asper (Terciopelo) venom is a lysine-49 phospholipase A2.

A basic, dimeric myotoxic protein, myotoxin II, purified from Bothrops asper venom has a similar molecular weight and is immunologically cross-reactive with antibodies raised to previously isolated B. asper phospholipases A2, except that it shows only 0.1% of the phospholipase activity against L-alpha-phosphatidylcholine in the presence of Triton X-100. Its 121 amino acid sequence, determined by automated Edman degradation, clearly identifies it as a Lys-49 phospholipase A2. Key amino acid differences between myotoxin II and phospholipase active proteins in the Ca2(+)-binding loop region, include Lys for Asp-49, Asn for Tyr-28, and Leu for Gly-32. The latter substitution has not previously been seen in Lys-49 proteins. Other substitutions near the amino terminus (Leu for Phe-5 and Gln for several different amino acids at position 11) may prove useful for identifying other Lys-49 proteins in viperid and crotalid venoms. Myotoxin II shows greater sequence identity with other Lys-49 proteins from different snake venoms (Agkistrodon piscivorus piscivorus, Bothrops atrox, and Trimeresurus flavoviridis) than with another phospholipase A2 active Asp-49 molecule isolated from the same B. asper venom. This work demonstrates that phospholipase activity per se, is not required in phospholipase molecules for either myotoxicity or edema inducing activities.

Amino Acid Sequence↗
PubMed1991

New toxins from the venom of the common tiger snake (Notechis scutatus scutatus).

Scutoxin A and B represent two isoforms of a new toxic protein from the venom of the Australian tiger snake, Notechis scutatus scutatus. Both isoforms, of apparent mol. wt 13,000, are less basic than either notexin or notechis II-5. They both have similar i.v. LD50-values in mice of ca 0.006 micrograms/g, and phospholipase activities of about 136 mumoles of fatty acid released/min/mg at 37 degrees C when acting on phosphatidylcholine in the presence of Triton X-100. Toxicities of the scutoxins are the same as notexin and about seven times more potent than notechis II-5. ELISAs and western blot analyses indicate that the new toxins are immunologically similar to notexin and notechis II-5, with phospholipase activities falling between these latter two proteins. When crude venom is initially passed over a gel filtration column, each scutoxin isoform co-elutes in a different fraction with notexin. Gel filtration experiments using purified samples of notexin and scutoxin have failed to demonstrate any evidence for the formation of higher mol. wt protein complexes. Peptide mapping suggests the presence of five glutamate residues in one of the protein isoforms. These findings, together with the high toxicity and active phospholipase levels, demonstrate that the new proteins are not the previously reported non-toxic and enzymatically inactive notechis II-1. The combination of gel filtration on Sephacryl S-200 and cation-exchange chromatography used to isolate the scutoxins also permits recovery of notexin and notechis II-5 in high purity.

Animals↗
PubMed1991

Specific binding of crotoxin to brain synaptosomes and synaptosomal membranes.

Crotoxin, the presynaptic neurotoxin from Crotalus durissus terrificus, was iodinated and used to demonstrate high affinity, specific binding to guinea-pig (Cavia porcellus) brain synaptosomes and synaptosomal membrane fragments. 125I-crotoxin binding to the membrane fragments displays two binding plateaus, (Kd1 = 4 nM and Kd2 = 87 nM, Bmax1 = 2 and Bmax2 = 4 pmoles/mg membrane protein), but binding to whole synaptosomes revealed only one plateau (Kd = 2 nM and Bmax = 5 pmoles/mg membrane protein). Rosenthal analyses of Scatchard plots yielded similar binding constants in the presence or absence of 0.025% Triton X-100. In addition to equilibrium analyses, kinetic analyses of 125I-crotoxin binding to synaptosomal membrane fragments gave a Kd-value of 3 nM. The Kd value was not significantly changed by the exclusion of added calcium, but the binding site number was lowered. Crotoxin binding was inhibited by the acidic subunit of crotoxin and several presynaptic neurotoxins, which were classified according to their inhibitory properties as, strong (acidic subunit of crotoxin, Mojave toxin, concolor toxin, taipoxin and pseudexin), moderate (ammodytoxin A and textilotoxin), weak (notexin and scutoxin A), very weak (notechis II-5) and non-inhibitory (basic subunit of crotoxin, beta-bungarotoxin, Crotalus atrox and porcine pancreatic phospholipases A2, dendrotoxin, and notechis III-4). Purified acidic subunit of crotoxin, the most potent competitor of crotoxin binding, was somewhat more competitive than intact crotoxin and the other strong inhibitors on a molar basis. Strong, moderate and weak inhibitor groups each differed from the preceding group by requiring about a ten fold increase in concentration to effect a 50% inhibition of crotoxin binding. The weak group was therefore at least two-orders of magnitude less effective than the strong inhibition shown by the acidic subunit of crotoxin. Treatment of synaptosomal membranes with protease K lowered 125I-crotoxin binding, whereas treatment with trypsin did not. Iodinated, phospholipase A2 from C. atrox venom showed no specific binding to whole synaptosomes. Our results demonstrate the presence and describe some of the properties of high affinity, specific binding sites in brain tissue for crotoxin and related presynaptic neurotoxins.

Animals↗