Identification and characterization of phospholipases A from the skin secretion of Pithecopus azureus anuran.

The present work reports the isolation, characterization and the complete sequence of a phospholipase A (PLA) present in the skin secretion of Pithecopus azureus. Among several peptides and small proteins previously described by our group from some species belonging to this amphibian genus (formerly named Phyllomedusa), a 15 kDa N-glycosylated protein showing PLA activity was purified, assayed, sequenced and named Pa-PLA. The Pithecopus azureus skin phospholipase A polypeptide chain is composed by 125 amino acid residues linked by seven disulfide bonds and two N-glycosylated sites (N67 and N108). The Pa-PLA enzymatic activity was qualitatively evaluated and compared to classical viperid PLA showing that both, native and deglycosylated Pa-PLA forms, are catalytically functional. The tridimensional molecular model of Pa-PLA indicates that the observed glycan moieties are suggestively placed far from the active site of that enzyme and therefore having little or no significant role on the direct interaction of the Pa-PLA catalytic pocket and its substrates.