title: Biochemical characterization of a phospholipase A2 from Photobacterium damselae subsp. piscicida.
abstract: Photobacterium damselae subsp. piscicida (Phdp) is the causative agent of fish photobacteriosis (pasteurellosis) in cultured cobia (Rachycentron canadum) in Taiwan. A component was purified from the extracellular products (ECP) of the bacterium strain 9205 by fast protein liquid chromatography (FPLC) and identified as a phospholipase. An N-terminal sequence of 10 amino acid residues, QDQPNLDPGK, was determined by mass spectroscopy (MS) and found to be identical with that of another Phdp phospholipase (GenBank accession no. BAB85814) at positions 21 to 30. The corresponding gene sequence of the phospholipase (GenBank accession no. AB071137) was employed to design primers for amplification of the sequence by the polymerase chain reaction (PCR). The PCR products were transformed into Escherichia coli, and a recombinant protein product was obtained which was purified as a His-tag fusion protein by Ni-metal affinity chromatography. A single 43-kDa band was determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Phosphatidylcholine was degraded by this protein to lysophosphatidylcholine and a fatty acid. These products were characterized by thin-layer (TLC) and gas chromatography (GC), respectively, allowing the identification of the protein as a phospholipase A2. The recombinant protein had maximum enzymatic activity between pH 4 and 7, and at 40 degrees C. The activity was inhibited by Zn(2+) and Cu(2+), activated by Ca(2+) and Mg(2+), and completely inactivated by dexamethasone and p-bromophenacyl bromide. A rabbit antiserum against the recombinant protein neutralized the phospholipase A2 activity in the ECP of Phdp strain 9205 and the recombinant protein itself. The recombinant protein was toxic to cobia of about 5 g weight with an LD50 value between 2 and 4 microg protein/g fish. The results revealed phospholipase A2 as a fish toxin in the ECP of Phdp strain 9205.
authors.affiliation: Department of Aquaculture, National Taiwan Ocean University, 2, Pei-Ning Road, Keelung, Taiwan.
authors.forename: Po-Yuan
authors.initials: PY
authors.lastname: Hsu
authors.affiliation: Department of Aquaculture, National Taiwan Ocean University, 2, Pei-Ning Road, Keelung, Taiwan.
authors.forename: Kuo-Kau
authors.initials: KK
authors.lastname: Lee
authors.affiliation: Eastern Marine Biology Research Center, Fisheries Research Institute, 22, Wuquan Road, Chenggong Township, Taitung, Taiwan.
authors.forename: Pei-Shan
authors.initials: PS
authors.lastname: Lee
authors.affiliation: Department of Aquaculture, National Taiwan Ocean University, 2, Pei-Ning Road, Keelung, Taiwan.
authors.forename: Chih-Chuang
authors.initials: CC
authors.lastname: Hu
authors.affiliation: Department of Aquaculture, National Taiwan Ocean University, 2, Pei-Ning Road, Keelung, Taiwan.
authors.forename: Cheng-Hui
authors.initials: CH
authors.lastname: Lin
authors.affiliation: Department of Aquaculture, National Taiwan Ocean University, 2, Pei-Ning Road, Keelung, Taiwan.
authors.forename: Ping-Chung
authors.initials: PC
authors.lastname: Liu
mesh_terms: D000595:Amino Acid Sequence; D001483:Base Sequence; D015153:Blotting, Western; D002849:Chromatography, Gas; D002855:Chromatography, Thin Layer; D017931:DNA Primers; D004591:Electrophoresis, Polyacrylamide Gel; D013058:Mass Spectrometry; D054467:Phospholipases A2 / Q000737:chemistry / Q000378:metabolism*; D010776:Photobacterium / Q000201:enzymology*; D016133:Polymerase Chain Reaction
chemical_list: D017931:DNA Primers; D054467:Phospholipases A2