Giant hornet (Vespa mandarinia) venomous phospholipases. The purification, characterization and inhibitory properties by biscoclaurine alkaloids.
Two species of giant hornet phospholipase B (PLB), alpha and beta, 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 alpha and beta were 29.5 and 26.0 kDa, respectively. The isoelectric point of alpha and beta enzymes were pH 10.6 and 10.7, respectively. The temperature optimum for egg yolk lecithin was a broad peak at 40-60 degrees 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 alpha and beta enzymes were 8.29 and 7.53 mg/ml for egg yolk lecithin, respectively. In the catalytic specificity for L-alpha-phosphatidylcholine-beta-oleoil-gamma-palmitoil, the K(m) values of alpha enzyme for gamma-palmitoil and beta-oleoil residues were 0.528 and 1.392 mM, respectively. While the K(m) values of beta enzyme for gamma-palmitoil and beta-oleoil residues were 7.91 and 2. 68 mM, respectively. Both alpha and beta enzymes were inhibited strongly by cepharanthine. The lecithin hydrolysis of alpha enzyme was competitively inhibited, but beta enzyme was uncompetitive. Cepharanthine also inhibited noncompetitively PLA(2)s of bovine pancreas, bee venom and Naja mossambica mossambica.
Abe
T
T
Hornet Researach Group, The Institute of Physical and Chemical Research, Saitama, Japan. abe@postman.rken.go.jp
Sugita
M
M
Fujikura
T
T
Hiyoshi
J
J
Akasu
M
M
D000470:Alkaloids / Q000494:pharmacology*; D000596:Amino Acids / Q000032:analysis; D000818:Animals; D001514:Bee Venoms / Q000201:enzymology*; D044182:Benzylisoquinolines; D002850:Chromatography, Gel; D004591:Electrophoresis, Polyacrylamide Gel; D004791:Enzyme Inhibitors / Q000494:pharmacology*; D008401:Gas Chromatography-Mass Spectrometry; D008245:Lysophospholipase / Q000037:antagonists & inhibitors / Q000302:isolation & purification*; D010713:Phosphatidylcholines / Q000378:metabolism; D014863:Wasps; D052117:Benzodioxoles
D000470:Alkaloids; D000596:Amino Acids; D001514:Bee Venoms; D044182:Benzylisoquinolines; D004791:Enzyme Inhibitors; D008245:Lysophospholipase; D010713:Phosphatidylcholines; C006947:cepharanthine; D052117:Benzodioxoles