Giant hornet ( Vespa mandarinia) venomous phospholipases : The purification, characterization and inhibitory properties by biscoclaurine alkaloids

Abstract

Two species of giant hornet phospholipase B (PLB), α and β, were purified from the venom of Vespa mandarinia. The purification procedure was simplified by two steps of column chromatographies, Sephadex G-100 and SP-Sepharose. The molecular sizes of PLB α and β were 29.5 and 26.0 kDa, respectively. The isoelectric point of α and β enzymes were pH 10.6 and 10.7, respectively. The temperature optimum for egg yolk lecithin was a broad peak at 40–60°C for both enzymes. Amino acid compositions of both enzymes were high contents of aspartic acid, glycine, leucine, lysine and other aliphatic amino acids. Cystine was similar amounts to other species of phospholipases (PLs). The K m values of α and β enzymes were 8.29 and 7.53 mg/ml for egg yolk lecithin, respectively. In the catalytic specificity for L-α-phosphatidylcholine-β-oleoil-γ-palmitoil, the K m values of α enzyme for γ-palmitoil and β-oleoil residues were 0.528 and 1.392 mM, respectively. While the K m values of β enzyme for γ-palmitoil and β-oleoil residues were 7.91 and 2.68 mM, respectively. Both α and β enzymes were inhibited strongly by cepharanthine. The lecithin hydrolysis of α enzyme was competitively inhibited, but β enzyme was uncompetitive. Cepharanthine also inhibited noncompetitively PLA 2s of bovine pancreas, bee venom and Naja mossambica mossambica.