Phospholipase A(2) with platelet aggregation inhibitor activity from Austrelaps superbus venom: Protein purification and cDNA cloning

Four phospholipase A(2) (PLA(2)) enzymes (Superbins a, b, c, and d) with va rying platelet aggregation inhibitor activities have been purified from Aus trelaps superbus by a combination of gel filtration, ion-exchange, and reve rsed-phase high-pressure liquid chromatography. Purity and homogeneity of t he superbins have been confirmed by high-performance capillary zone electro phoresis and mass spectrometry. The electron spray ionization mass spectrom etry data showed that their molecular masses range from 13,140 to 13,236 Da . Each of the proteins has been found to be basic and exhibit varying degre es of PLA(2) activity. They also displayed different platelet aggregation i nhibitory activities. Superbin a was found to possess the most potent inhib itory activity with an IC50 of 9.0 nM, whereas Superbin d was found to be l east effective with an IC50 of 3.0 mu M. Superbins b and c were moderately effective with IC50 values of 0.05 and 0.5 mu M, respectively. The amino-te rminal sequencing confirmed the identity of these superbins. cDNA cloning r esulted in the identification of 17 more PLA(2) isoforms in A. superbus ven om. It has also provided complete information on the precursor PLA(2). The precursor PLA(2) contained a 27-amino-acid signal peptide and 117- Ito 125- amino-acid PLA(2) (molecular mass ranging from 13,000 to 14,000 Da). Two of these PLA(2) enzymes resembled more closely (87%) Superbin a in structure. Two unique PLA(2) enzymes containing an extra pancreatic loop also have be en identified among the isoforms. (C) 2000 Academic Press.