Cloning, expression and biochemical characterization of a basic-acidic hybrid phospholipase A(2)-II from Agkistrodon halys Pallas

A cDNA encoding a basic-acidic hybrid phospholipase A(2)-II from Agkistrodo n halys Pallas with an N-terminus highly homologous to that of BPLA(2) and a C-terminus sequence almost the same as that of APLA(2) was inserted into a bacterial expression vector and effectively expressed in Escherichia coli RR1. The protein was produced as insoluble inclusion bodies. After partial purification by washing, the inclusion bodies with Triton X-100, denaturin g and refolding, the renatured recombinant protein was purified by FPLC col umn superose 12. The purified recombinant enzyme with an isoelectric point of pH 6.8 could cross-react with antiserum prepared against acidic phosphol ipase A(2). The enzymatic activity of the expressed basic-acidic hybrid pho spholipase A(2)-II is close to that of denatured-refolded native basic phos pholipase A(2), and has the same inhibiting effect on platelet aggregation as denatured-refolded acidic phospholipase A(2), but lacks the hemolytic ac tivity of denatured-refolded basic phospholipase A(2) To study the structur al relationships among basic phospholipase A(2), acidic phospholipase A(2) and basic-acidic hybrid phospholipase A(2)-II, molecular modeling of basic- acidic hybrid phospholipase A(2)-II was dose. The roles of various amino ac id residues in the enzymatic activity and pharmacological activities of pho spholipase A(2) are discussed. (C) 1999 Elsevier Science B.V. All rights re served.