Crystallization and preliminary diffraction analysis of caudoxin and notexin; two monomeric phospholipase A2 neurotoxins.

Two monomeric neurotoxic phospholipases A2 have been crystallized and their diffraction properties characterized. Crystals of caudoxin (from the venom of Bitis caudalis) and notexin (from the venom of Notechis scutatus scutatus) were grown at neutral pH, in the absence of calcium ion, and diffract to a resolution of 2.3 and 1.6 A, respectively.

A steady-state kinetic analysis of the reaction between arginine esterase E-I from Bitis gabonica venom and synthetic arginine substrates and the influence of pH, temperature and solvent deuterium isotope.

1. Using synthetic arginines as substrates, steady-state kinetic studies showed a deviation from Michaelis-Menten kinetics for esterase E-I purified from the venom of Bitis gabonica. Graphical analysis indicated a rate equation of at least a degree of 3:3. 2. pH variation of the kinetic parameters indicated the involvement of groups with pK values of approximately 7 and approximately 9 which had to be in the ionic form for activity. 3. Solvent isotope studies suggested transition states where proton transfer or reorganization was the rate-limiting step of proteolytic catalysis. A single protogenic site was postulated. 4. Temperature effects on the enzymic reaction showed a significant reduction in entropy loss upon formation of the transition state with both esters and extended tail polypeptide-anilides in comparison with the activation entropy for benzoyl-L-arginine p-nitroanilide.

Inactivation of arginine esterase E-I of Bitis gabonica venom by irreversible inhibitors including a water-soluble carbodiimide, a chloromethyl ketone and isatoic anhydride.

1. Esterase E-I from Bitis gabonica was inactivated with irreversible inhibitors which included studies with a water-soluble carbodiimide, an affinity labelling peptide and a mechanism-based inactivator. 2. The reaction with 1-ethyl-3(3-dimethylaminopropyl)-carbodiimide was biphasic and the dominant part followed saturation kinetics. At pH 5.5 a rate constant of 0.4 min-1 for inactive enzyme formation was calculated and a dissociation constant (Ki) of 0.2 M for the enzyme-inhibitor complex. 3. Inactivation with D-Phe-Pro-Arg-chloromethyl ketone indicated a two-step mechanism, for which the reaction parameters at pH 8.0 were determined. The Ki value was 0.2 microM and the inactivation rate was 2.5 min-1. 4. With isatoic anhydride pseudo-first-order kinetics was observed. At pH 8.0 a rate constant of 0.9 min-1 and a Ki of 2.0 mM were obtained. The inactivation of the enzyme was found to be governed by a group in the enzyme showing a pK value of 7.3.

Deviation from Michaelis-Menten kinetics for esterase E-II from the venom of Bitis gabonica, using synthetic arginine amides as substrates.

Steady state kinetic studies on the reaction between esterase E-II from the venom of Bitis gabonica and the fluorogenic substrates, N-alpha-benzoyl-L-phenylalanyl-L-valyl-L-arginine-4-methylcoumaryl-7-amide and N-alpha-benzoyl-L-arginine-4-methylcoumaryl-7-amide were found to deviate from Michaelis-Menten kinetics. Analysis of algebraic graphs and application of non-linear regression allowed an empirical rate law to be selected. The results revealed a rate equation of at least third degree at pH values above 7.0 and of 2:2 degree in the pH range 6-7. The data were interpreted in terms of a molecular model involving an enzyme with one catalytic site and several auxiliary or regulatory sites which, through cooperative effects, may either activate or inhibit the enzyme. Substrate activation is observed at low substrate values and might follow from an obligatory order of binding involving two of the sites, the modifier substrate molecule binding before the substrate molecule undergoing transformation to products. Inhibitory sites apparently become available only at alkaline pH. The inhibition is only noted at high substrate concentrations and is of the partial type.

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.

A new type of toxin in the venom of snakes of the genus Atractaspis (Atractaspidinae).

The venom of Atractaspis is unique in having a large percentage of both high and low molecular weight components. Its Sephadex G-50 S5 fraction appears to represent a new type of toxin that contains 17-18 Asx, 13-14 Cys and 10-11 Glx out of a total of 72-78 amino acids. The N-terminal of this toxin does not seem to resemble any of the known toxins. The overall lethal potency of the venom is very high; i.v. injections of 5 microgram of venom or 1 microgram of fractions S5 or S6 per mouse causes death within minutes. The results of the present study corroborate previous findings that suggested a separate grouping of the snakes genus Atractaspis at the subfamilial or familial level.

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.

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.

A steady state analysis of pH and temperature effects on the action of an arginine esterase from the venom of the gabon adder, Bitis gabonica, on synthetic arginine substrate.

The influence of pH and temperature on the kinetic properties of esterase E-II from Bitis gabonica venom has been studied. The pH profiles for the hydrolysis of N alpha-benzoyl-L-arginine ethyl ester showed that a group with pK approximately 7 must be ionized for activity. Measurement of N alpha-benzyl-L-arginine-p-nitroanilide hydrolysis reveals that the pK of the active site is significantly lowered (i.e. to approximately 6.3) in the enzyme-substrate complex. The temperature effect on the pK suggests the presence of a carboxylate in the active site of the enzyme. This suggestion is corroborated by the influence of organic solvent perturbations of the pK indicating a catalytic group of the neutral acid type. Values for the thermodynamic parameters associated with activation are reported. The transition state for ester degradation is at lower delta G and delta H levels than that for amide degradation. delta H and delta S for ester hydrolysis were noted to compensate each other with a compensation temperature Tc = 339K. Compensation can probably be related to weak bonding effects.

Isolation and characterization of three lutropin isohormones from the pituitary gland of the ostrich (Struthio camelus).

Avian lutropin has been isolated from pituitary glands of the ostrich (Struthio camelus) in three homogeneous forms (designated isohormones). The different homogeneous forms of ostrich lutropin were characterized physically and chemically in terms of molecular weight, electrophoretic mobility, isoelectric points, amino acid and carbohydrate composition. From these characteristics it was evident that the isohormones are very similar. The differences between these isohormones can be attributed to differences in carbohydrate composition, especially sialic acid.

A kinetic analysis of the hydrolysis of synthetic arginine substrates by arginine esterases from the venom of the gabooon adder, Bitis gabonica.

The kinetics of arginine esterases E-I, E-II and E-III from the venom of Bitis gabonica were investigated. With N alpha-benzoyl-L-arginine ethyl ester as substrate linear competitive inhibition versus L-arginine was observed while ethanol gave rise to S-parabolic I-linear noncompetitive inhibition. Hydrolysis of N alpha-benzoyl-L-arginine-p-nitroanilide was noncompetitively inhibited by p-nitroaniline. Both slopes and intercepts of double reciprocal plots were a linear function of inhibitor concentration. Ethanol gave complex inhibition kinetics which could be interpreted in terms of mixed dead-end and alternate product inhibition (S-parabolic I-hyperbolic noncompetitive inhibition). These results imply an ordered uni-bi as the minimal kinetic mechanism wherein ethanol (or amine when amide is used as substrate) is released first from the enzyme surface, followed by the liberation of arginine. The enzymes are inactivated by phenylmethane sulfonyl fluoride which suggests the presence of an essential serine in the active sites of the enzymes. The enzymes may therefore be classified in the group of serine proteases.

Snake venom. The amino-acid sequence of the subunits of two reduced and S-carboxymethylated proteins (C8S2 and C9S3) from Dendroaspis angusticeps venom.

Two proteins (C8S2 and C9S3) were purified from Dendroaspis angusticeps venom. Whereas protein C8S2 comprises 124 amino acid residues, protein C9S3 contains 125 and each includes 16 half-cystines. After reduction and S-carboxymethylation of the proteins, the subunits were shown to contain 62--63 residues including 8 half-cystine residues. The complete sequences of the subunits have been elucidated. The sequences of the subunits of protein C8S2 and C9S3 are homologous to those of protein S2C4 from D. jamesoni kaimosae. Further, in all the subunits one of the structurally invariant amino acids, Tyr25, has been replaced by phenylalanine or asparagine and the Cys66 occurs in position 57. All the subunits apparently contain only two of the five functionally invariant residues of the neurotoxins, viz. Lys27 and Trp29. Mixtures of proteins S2C4, C8S2, C9S3 and angusticeps-type proteins showed a marked synergistic toxic effect.

Histidine and lysine residues and the activity of phospholipase A2 from the venom of Bitis gabonica.

Chemical modification of phospholipase A2 (phosphatide 2-acyl-hydrolase, EC 3.1.1.4) from the venom of gaboon adder (Bitis gabonica) showed that histidine and lysine residues are essential for enzyme activity. Treatment with p-bromophenacyl bromide or pyridoxal 5'-phosphate resulted in the specific covalent modification of one histidine or a total of one lysine residue per molecule of enzyme, respectively, with a concomitant loss of enzyme activity. Competitive protection against modification and inactivation was afforded by the presence of Ca2+ and/or micellar concentrations of substrate analogue, lysophosphatidylcholine. Neither modification caused any significant conformational change, as judged from circular dichroic properties. Amino acid analyses and the alignment of peptides from cyanogen bromide and proteolytic cleavage of modified enzyme preparations delineated His-45 as the only residue modified by p-bromophenacyl bromide. However, pyridoxal 5'-phosphate was shown to have reacted not with a single lysine but with four different ones (residues 11, 33, 58 and 111) in such a manner that an overall stoichiometry of one modified lysine residue/molecule enzyme resulted. Apparently, the essential function of lysine could be fulfilled by any one out of these four residues.

The amino acid sequence of three non-curarimimetictoxins 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.