title: Purification, characterization and molecular cloning of alpha-2-macroglobulin in cobia, Rachycentron canadum.
abstract: Alpha-2-macroglobulin (α-2-M) is a broad spectrum protease inhibitor which is abundant in the plasma of vertebrates and several invertebrates. The α-2-M was purified from cobia (Rachycentron canadum) plasma by a four-step procedure: poly ethylene glycol fractionation, affinity chromatography, hydrophobic interaction chromatography and ion exchange chromatography on Fast Protein liquid chromatography system in the present study. It migrated as one protein band with a molecular mass of about 360 kDa in the native state, whereas in SDS-PAGE it was about 180 kDa under non-reducing condition. This result revealed that the native protein was a dimer. In addition, it was cleaved into two different fragments of molecular mass about 93 and 87 kDa when reduced by dithiothreitol (DTT). The anti-protease activity of the purified α-2-M was apparently decreased as temperature elevated above 50 °C. The α-2-M exhibited highest protease inhibitory activity at pH 9. The results indicate that the α-2-M is a heat-labile and alkaline protease inhibitor. The purified α-2-M exhibited more than 50% protease inhibitory activity against extracellular products (ECP) of Vibrio alginolytius isolated from diseased cobia. It seems that the protease activities in ECP may be affected by the plasma α-2-M. The protease inhibitory activities of cobia plasma or purified α-2-M were decreased when incubated with 10 mM methylamine for 30 min. The α-2-M cDNA consisted of 4611 bp with an open reading frame of 4374 bp had been cloned from cobia liver. This sequence contained thioester domain (GCGEQ) and thirteen predicted N-linked glycosylation sites. In addition, the amino acid sequence of thioester domain and genes of adjacent regions of cobia α-2-M were further compared with sequences of known fish species in GenBank. The unweighted pair group method using arithmetic average (UPGMA) was employed to construct the phylogenetic trees of α-2-M among different fish species (freshwater fish, sea water fish and primitive fish), and all these fish species were then clustered into three groups. The cobia α-2-M was closer to that of sea water fish than that of freshwater fish compared basing on its similarity of amino acid sequence and phylogenetic analysis of the partial gene.
authors.affiliation: Department of Aquaculture, National Taiwan Ocean University, 2, Pei-Ning Road, Keelung 202, Taiwan.
authors.forename: Wen-Hsiao
authors.initials: WH
authors.lastname: Chuang
authors.affiliation: Department of Aquaculture, National Taiwan Ocean University, 2, Pei-Ning Road, Keelung 202, Taiwan.
authors.forename: Ping-Chung
authors.initials: PC
authors.lastname: Liu
authors.affiliation: Department of Aquaculture, National Taiwan Ocean University, 2, Pei-Ning Road, Keelung 202, Taiwan.
authors.forename: Chia-Yu
authors.initials: CY
authors.lastname: Hung
authors.affiliation: Department of Aquaculture, National Taiwan Ocean University, 2, Pei-Ning Road, Keelung 202, Taiwan. Electronic address: b0092@mail.ntou.edu.tw.
authors.forename: Kuo-Kau
authors.initials: KK
authors.lastname: Lee
mesh_terms: D000818:Animals; D002846:Chromatography, Affinity; D002852:Chromatography, Ion Exchange; D002853:Chromatography, Liquid; D003001:Cloning, Molecular; D019295:Computational Biology; D017931:DNA Primers / Q000235:genetics; D018076:DNA, Complementary / Q000235:genetics; D004591:Electrophoresis, Polyacrylamide Gel; D006863:Hydrogen-Ion Concentration; D008744:Methylamines / Q000378:metabolism; D010473:Perciformes / Q000235:genetics*; D011092:Polyethylene Glycols; D011480:Protease Inhibitors / Q000097:blood / Q000302:isolation & purification*; D013696:Temperature; D000511:alpha-Macroglobulins / Q000235:genetics* / Q000302:isolation & purification
chemical_list: D017931:DNA Primers; D018076:DNA, Complementary; D008744:Methylamines; D011480:Protease Inhibitors; D000511:alpha-Macroglobulins; D011092:Polyethylene Glycols; C027451:methylamine
keywords: Alpha-2-macroglobulin; Extracellular products; Plasma; Protease inhibitory activity; Rachycentron canadum