Cloning, expression and biochemical characterization of a basic-acidic hybrid phospholipase A2-II from Agkistrodon halys pallas.
A cDNA encoding a basic-acidic hybrid phospholipase A2-II from Agkistrodon halys Pallas with an N-terminus highly homologous to that of BPLA2 and a C-terminus sequence almost the same as that of APLA2 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, denaturing and refolding, the renatured recombinant protein was purified by FPLC column superose 12. The purified recombinant enzyme with an isoelectric point of pH 6.8 could cross-react with antiserum prepared against acidic phospholipase A2. The enzymatic activity of the expressed basic-acidic hybrid phospholipase A2-II is close to that of denatured-refolded native basic phospholipase A2, and has the same inhibiting effect on platelet aggregation as denatured-refolded acidic phospholipase A2, but lacks the hemolytic activity of denatured-refolded basic phospholipase A2. To study the structural relationships among basic phospholipase A2, acidic phospholipase A2 and basic-acidic hybrid phospholipase A2-II, molecular modeling of basic-acidic hybrid phospholipase A2-II was done. The roles of various amino acid residues in the enzymatic activity and pharmacological activities of phospholipase A2 are discussed.
Liu
X
X
Shanghai Institute of Biochemistry, Chinese Academy of Science, Shanghai 200031, China.
Pan
H
H
Yang
G
G
Wu
X
X
Zhou
Y
Y
D017836:Agkistrodon; D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D003001:Cloning, Molecular; D003435:Crotalid Venoms / Q000201:enzymology*; D018076:DNA, Complementary / Q000096:biosynthesis / Q000737:chemistry; D054501:Group II Phospholipases A2; D008969:Molecular Sequence Data; D010741:Phospholipases A / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics*; D054467:Phospholipases A2
D003435:Crotalid Venoms; D018076:DNA, Complementary; D010741:Phospholipases A; D054501:Group II Phospholipases A2; D054467:Phospholipases A2