Purification and characterization of five variants of phospholipase A2 and complete primary structure of the main phospholipase A2 variant in Heloderma suspectum (Gila Monster) venom

1 Five increasingly anionic variants (Pa1–Pa5) of Ca2+‐dependent phospholipase A2 were purified to homogeneity from the venom of the lizard Heloderma suspectum (Gila monster). The purification procedure was based on semi‐preparative reverse‐phase HPLC followed by anion‐exchange HPLC and analytical reverse‐phase HPLC. 2 Their Mr were 17000–18000, as deduced by SDS/PAGE. Specific activities tested by the capacity to hydrolyze phosphatidylcholines at pH 8.5 decreased as follows: Pa3 > Pa5 > Pa4 > Pa1 > Pa2. These activities showed the same optimum pH (9.0), were mainly of the phospholipase A2 type and were lost upon p‐bromophenacyl bromide treatment. 3 All five phospholipases efficiently stimulated amylase release from dispersed rat pancreatic acini at pH 7.4, their potency decreasing as follows: Pa2 > Pa1∼ Pa4 > Pa3∼ Pa5. No deleterious effect was apparent based on the lack of lactate dehydrogenase release. 4 The five variants, Pa1–Pa5, differed significantly in amino acid composition and this, together with distinct antigenic properties of Pa2 and Pa5, establishes the subheterogeneity of this new type of phospholipase A2, despite the fact that the N‐terminal amino acid sequence (31 residues) of Pa1–Pa5 was exactly the same. 5 The full sequence of the major variant, Pa5, showed that this 142‐amino‐acid protein exhibited greater similarity to the bee venom enzyme than to any class I or class II secretory phospholipase A2 from snake venom and mammalian pancreas. While Pa5 displayed the highly conserved region between Asp30 and Cys39 (the essential active site of all phospholipases A2), its salient original points included 10 half‐cystine residues only, an incomplete N‐terminal sequence, large changes in the putative calcium loop, several alterations after the active site and a C‐terminal extension never seen in other phospholipases A2, with the only exception being bee venom.