The amino acid sequence of a non-toxic phospholipase AZ, ammodytin I,, from the venom of the long-nosed viper (Vipera ammodytes ammodytes) and its cDNA sequence have been determined. The protein sequence was elucidated by sequencing the peptides generated by CNBr cleavage, mild acid hydrolysis and tryptic digestion of maleylated and non-maleylated protein. Sequencing of the cDNA showed that the protein is synthesized as an 137-amino-acid-residue precursor molecule consisting of a 16-residue signal peptide, followed by a 121-residue mature enzyme. Ammodytin I2 cDNA shows 73% nucleotide and 59% amino acid identities in the mature protein region in comparison to that of ammodytoxin A, the most presynaptically neurotoxic phospholipase A, from the long-nosed viper. Identities in the signal-peptide region are considerably higher, 96% and loo%, respectively. Snake venoms are a rich source of phospholipases A, (PLA,). From the venom of the long-nosed viper (Vipera ammodytes ammodytes), six PLA2 species and their homologues have been isolated [l]. Three of them, ammodytoxins A, B and C, are basic proteins with strong presynaptic neurotoxicity. All three toxins were sequenced [2 -41. The fourth and most basic protein of the venom, ammodytin L, presumably devoid of PLA2 activity, has a myotoxic action [5]. The neutral PLA, from the venom was designated as venom fraction I [l]. It was the most enzymatically active and non-toxic [ 11.Snake PLA2 are an intriguing group of proteins. In addition to enzymatic activity, many of them show different pharmacological effects such as neurotoxic, myonecrotic, cardiotoxic, hemolytic, anticoagulant, edema-inducing, convulsant or hypotensive action [6]. Such an extensive functional diversity within this group of structurally similar proteins raised the question of the relationship between their structure and their pharmacological effects. Although a considerable number of PLAz have already been sequenced, a clear answer has not yet been found. Information about the primary structure of another PLA2, ammodytin I,, should therefore provide some new data in the ongoing analysis of the toxic site.In this paper, we report the complete amino acid sequence of ammodytin I,, together with its cDNA sequence.
MATERIALS AND METHODS
MaterialsAmmodytin I, was isolated from venom fraction I [l], which was found, by isoelectric focusing, to be composed of two phospholipase-A,-active bands. The separation of crude venom was performed on carboxymethyl-cellulose (CM-cellulose) at pH 8.2 in 0.05 M ammonium acetate [l], where nontoxic enzyme eluted before the application of the salt gradient. In the next step, high molecular mass components were removed by Sephadex G-100 chromatography in 0.35 M 8-alanine, pH 4.4. Finally, the two non-toxic enzymes were separated on CM-cellulose at pH 7.2 in 0.1 M Tris/HCl, containing 0.02 M CaCl,, and designated as ammodytin I1 and ammodytin 12. The latter was 2-3 times more abundant in different batches of the venom. [3H]iodoacetic acid was obtained from Amersham. CNBr and ...