Cornelis C. Viljoen

The structure of cyanoginosin-LA, a cyclic heptapeptide toxin from the cyanobacterium Microcystis aeruginosa

The structure of cyanoginosin-LA (previously referred to by us as toxin BE-4) is cyclo(-D-Ala-L-Leu-erythro-β-methyl-D-isoAsp-L-Ala-Adda-D-Glu-N-methyldehydroAla)(17a), where Adda refers to the novel β-amino acid residue of 3-amino-9-methoxy-2,6,8-trimethyl-10-phenyldeca-4,6-dienoic acid (11). The connectivity of Adda is deduced from 1H- and 13C-n.m.r. data, but the stereochemistry at carbons 2,3,8, and 9 remains unknown. The residue sequence is derived from the mass spectra of cyclic and linear derivatives of the toxin. A new nomenclature for the related toxins of Microcystis aeruginosa is discussed.

Isolation and characterization of four toxins from the blue-green alga, Microcystis aeruginosa

Two alternative procedures for the isolation of toxins from the blue-green alga, Microcystis aeruginosa forma aeruginosa, are described. A novel approach is reported, whereby contaminating impurities are succinylated, exploiting the absence of free amino groups in toxin variants. All toxin variants comprise a hydrocarbon blocking group, five amino acid residues detectable by conventional means, while methylamine is liberated upon acid hydrolysis. Possible structural features are discussed relating to the observed chemical and physical properties of the toxins.

Configuration assignments of the amino acid residues and the presence of N-methyldehydroalanine in toxins from the blue-green alga, Microcystis aeruginosa

The configuration assignment of the alpha-carbon atom of amino acid residues in four toxin variants from Microcystis aeruginosa have been made by stereospecific enzymic transformations. The relative conformation assignment of the beta-carbon atom of beta-CH3-aspartic acid could be made by comparison of the electrophoretic mobility with literature values reported for the authentic compound. The presence of an N-methyldehydroalanine residue, which, due to elimination of methylamine under hydrolytic conditions, previously escaped detection by conventional means, has been confirmed by identification of N-methylalanine in the hydrolysate after reduction of toxin with sodium borohydride.

Separation of Bitis gabonica (Gaboon adder) venom arginine esterases into kinin-releasing, clotting and fibrinolytic factors

Abstract C. C. Viljoen , C. M. Meehan and D. P. Botes . Separation of Bitis gabonica (Gaboon adder) venom arginine esterases into kinin-releasing, clotting and fibrinolytic factors. Toxicon17, 145–154, 1979. The arginine ester hydrolysing activity of the venom of Bitis gabonica has been isolated and purified by a combination of Sephadex gel filtration and ion-exchange chromatography on DEAE-cellulose. The esterases were designated E-I, E-II and E-III according to their sequence of elution from DEAE-cellulose. A molecular weight of about 32,000 was calculated for the enzymes which contain 20–25% by weight of carbohydrate. The enzymes appear to be specific for arginine esters, since lysine esters are not hydrolysed, and showed low proteolytic activity against casein. Distinct biological properties were found for the esterases, E-I, EII and E-III showing kinin-releasing, fibrinolytic and clotting activity, respectively.

Isolation and amino acid sequence of caudoxin, a presynaptic acting toxic phospholipase A2 from the venom of the horned puff adder (Bitis caudalis)

A presynaptic acting toxic phospholipase A2, designated caudoxin, was purified from the venom of Bitis caudalis by a combination of gel filtration and ion-exchange chromatography. The specificity of the enzyme was shown to be of the A2 type. The enzyme contains 121 amino acid residues in a single chain and is cross-linked by seven disulfide bridges. Application of cyanogen bromide cleavage and digestion with trypsin and chymotrypsin yielded peptides providing the necessary overlaps to complete derivation of the sequence. Structural features of caudoxin in relation to other toxic and non-toxic phospholipases A2 are discussed.

Purification of phospholipase A from Bitis gabonica venom

Abstract Bitis gabonica venom was separated on Sephadex G-50 and the major enzymes present in the venom localized by assaying the eluate against specific substrates. Phospholipase A was purified and its positional specificity towards phosphatidylcholine shown to be of the A 2 type. The amino acid composition of the purified phospholipase A is reported. An equilibrium is shown to exist between monomer and dimer which is favoured towards the dimer by addition of Ca 2+ and towards the monomer by EDTE.

The amino acid sequence of three non-curarimimetic toxins from Naja nivea venom☆

Three toxins of the non-curarimimetic type have been isolated from the venom of the Cape cobra Naja nivea. The basic and hydrophobic amino acids are dominant in all three toxins. They comprise 60 amino acid residues with 4 intrachain disulphide linkages. The toxins have been characterized with respect to their linear structures and immunochemical properties. Toxicity and hemolytic data suggest a much higher affinity for receptors on the heart cell membrane than for that of the red cell.

An essential tryptophan in the active site of phospholipase A2 from the venom of Bitis gabonica

The role of tryptophan in phospholipase A2 (EC 3.1.1.4) from the venom of the gaboon viper, Bitis gabonica, has been investigated. Modification of the enzyme with N-bromosuccinimide and 2-nitrophenylsulfenylchloride showed that the two tryptophan residues in the enzyme, viz. Trp-28 and Trp-59, differ in reactivity towards the reagents. Only Trp-28 reacted with N-bromosuccinimide while a preferential reaction occurred between Trp-59 and 2-nitrophenyl-sulfenylchloride. In each case it was found that loss of enzyme activity was specifically correlated with modification of TRP-28. CD spectra indicated that neither the local nor the gross conformation of the enzyme was altered by modification of Trp-28 and it was therefore concluded that Trp-28 is crucial for enzyme activity. The active enzyme was protected against N-bromosuccinimide inactivation by micellar concentrations of substrate or substrate analogue, suggesting that Trp-28 is involved in substrate binding.

Bitis gabonica venom: A kinetic analysis of the hydrolysis by phospholipase A2 of 1,2-dipalmitoyl-sn-glycero-3-phosphorylcholine

Abstract A kinetic study on the phospholipase As from Bitis gabonica venom, has been undertaken. The effects of substrate and Ca 2+ concentration and pH on the reaction rate have been evaluated. Initial velocity studies at pH 8.0 and 20° C, gave intersecting initial velocity patterns that conform to a mechanism in which Ca 2+ adds first and dipalmitoyllecithin second, the addition of Ca 2+ being at thermodynamic equilibrium. Product inhibition studies at pH 8.0 and 20° C accorded with a previously proposed mechanism and are consistent with a mechanism in which the products are released in an obligatory way. The results suggested fatty acid to be the first released product and lysolecithin the second released product. The effect of pH on the reaction rate indicated an optimum in the alkaline pH range.

Spectral properties of Bitis gabonica venom phospholipase A2 in the presence of divalent metal ion, substrate and hydrolysis products

Abstract Bitis gabonica phospholipase A 2 binds 2 moles of Ca 2+ per mole of dimer, giving rise to spectral perturbations of tryptophan chromophores in the enzyme. The spectral changes are taken to originate from both solvent and charge effects. A pH dependent perturbation is noted both in the presence and absence of Ca 2+ ; however, the character of the perturbation in absence of metal ions is different from the perturbation observed in the presence of Ca 2+ . The data suggest that Ca 2+ induces a conformational change in the enzyme, allowing the productive binding of substrate. A change in tryptophan absorption is also noted in the presence of dipalmitoyllecithin, lysolecithin and palmitic acid. However, perturbation by palmitic acid causes a blue shift of the spectrum as opposed to a red shift caused by lysolecithin and dipalmitoyllecithin. Fine structure in the 270–300 nm range due to tryptophan perturbation, is abolished when lysolecithin binds to the enzyme in the presence of Ca 2+ . This is interpreted to mean that the perturbable tryptophan is involved in substrate binding.