Discovery of serum L-amino acid oxidase in the rockfish Sebastes schlegeli: Isolation and biochemical characterization

• Published in: Comparative Biochemistry and Physiology Part B: Biochemistry and Molecular Biology Vol. 157; no. 4; pp. 351 - 356
• Main Authors: Kitani, Yoichiro, Ishida, Masami, Ishizaki, Shoichiro, Nagashima, Yuji
• Format: Journal Article
• Language: English
• Published: England Elsevier Inc 01-12-2010
• Subjects: Aeromonas hydrophila; Aeromonas salmonicida; Amino Acid Sequence; Animals; Anti-Bacterial Agents - blood; Anti-Bacterial Agents - pharmacology; Antibacterial protein; Fish Proteins - blood; Fish Proteins - chemistry; Fish Proteins - isolation & purification; Fishes - blood; Fishes - immunology; Hydrogen peroxide; Immunity, Innate; Innate immunity; L-Amino acid oxidase; L-Amino Acid Oxidase - blood; L-Amino Acid Oxidase - chemistry; L-Amino Acid Oxidase - isolation & purification; Marine; Molecular Sequence Data; Pleuronectiformes; Rockfish Sebastes schlegeli; Sebastes schlegeli; Serum; Serum; Rockfish Sebastes schlegeli; Innate immunity; Hydrogen peroxide; L-Amino acid oxidase; Antibacterial protein
• ISSN: 1096-4959; 1879-1107
• DOI: 10.1016/j.cbpb.2010.08.006

Fish have a complex innate defense mechanism against microbial invasion. In particular, epidermal mucus and serum in fish play important roles in innate immunity and contain a variety of bioactive substances such as complements, lectins and lysozymes, involved in host defense. Recently,
L-amino acid oxidases (LAOs) with antibacterial activity were isolated from the skin and/or gill mucous secretions of rockfish, great sculpin and flounder, and were identified to be a novel type of antibacterial protein in the integument of fish. In the present study, we found LAO activity in the serum of rockfish
Sebastes schlegeli. The LAO was isolated from the serum by sequential column chromatography of Con-A lectin affinity chromatography, anion exchange HPLC, hydroxyapatite HPLC and gel filtration HPLC, and characterized. The LAO (a molecular mass of 160
kDa) comprised subunits with a molecular mass of 53
kDa and showed strict substrate specificity, catalyzing only
L-lysine with
Km 0.37
mM and
kcat 57.1
s
−
1
. The serum LAO exhibited a broad antibacterial activity against Gram positive and negative bacteria, most potently against
Aeromonas hydrophila and
Aeromonas salmonicida with a minimum inhibitory concentration of 0.078
μg/mL. This is the first report of LAO in the serum of fish and its involvement in innate immunity in the rockfish body.