Graduate School of Fisheries Sciences, Hokkaido University, Hakodate, Hokkaido 041-8611, Japan
Site-directed mutagenesis study of phospholipase A2 (PLA2) from the pyloric ceca of starfish Asterina pectinifera was used to probe the relationship between polar-group specificity and structure of the pancreatic loop region. The sequence of the cDNA encoding the starfish PLA2 was exchanged by the oligonucleotide-directed dual amber-long and accurate polymerase chain reaction method to insert Lys residue between Cys 62 and Gly63 of the wild-type PLA2 (WT PLA2) expressed by Escherichia coli. The modified cDNA was inserted into the expression plasmid pET-16b, and PLA2 mutant was expressed in E. coli BL21 (DE3) by induction with isopropyl-β-D(−)-thiogalactopyranoside. The PLA2 mutant produced as inclusion bodies was dissociated with urea and 2-mercaptoethanol and renatured by dialyzing against Tris-HCl buffer. Renatured PLA2 mutant showed essentially the same properties as the WT PLA2 with respect to positional specificity of substrate, optimum pH and Ca2+ requirement. However, the PLA2 mutant hydrolyzed phosphatidylethanolamine (PE) more effectively than the WT PLA2. Therefore, it was suggested that the structure of the pancreatic loop region may be associated with polar-group specificity of PE.