Characterization of Vibrio mimicus phospholipase A (PhlA) and cytotoxicity on fish cell

Vibrio mimicus is a typical strain of Vibrio cholerae and produces a phospholipase (PhlA) which shares a highly conserved amino acid sequence with the lecithinase (Lec) of V. cholerae. The recombinant protein (rPhlA) produced from the phlA gene of V. mimicus was expressed in Escherichia coli as His-...

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Bibliographic Details
Published in:Biochemical and biophysical research communications 2002-10, Vol.298 (2), p.269-276
Main Authors: Lee, Jong-Hee, Ahn, Sun-Hee, Kim, Sun-Hoi, Choi, Yoon-Hyeok, Park, Kee-Jai, Kong, In-Soo
Format: Article
Language:English
Subjects:
Animals
Cell Line
Cytotoxicity
Escherichia coli - genetics
Fishes
Hemolysin Proteins - genetics
Hemolysin Proteins - metabolism
Hemolysin Proteins - pharmacology
Hemolytic activity
Hydrogen-Ion Concentration
Metals - pharmacology
phlA gene
Phospholipase A
Phospholipases A - genetics
Phospholipases A - metabolism
Phospholipases A - toxicity
Phospholipids - metabolism
Recombinant Fusion Proteins - isolation & purification
Substrate Specificity
Temperature
Vibrio - enzymology
Vibrio cholerae
Vibrio mimicus
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Summary:Vibrio mimicus is a typical strain of Vibrio cholerae and produces a phospholipase (PhlA) which shares a highly conserved amino acid sequence with the lecithinase (Lec) of V. cholerae. The recombinant protein (rPhlA) produced from the phlA gene of V. mimicus was expressed in Escherichia coli as His-tag fused protein. The rPhlA was purified by gel filtration and Ni–metal affinity chromatographies. When the action mode was investigated by TLC and GC–MS, the purified rPhlA protein showed a phospholipase A activity, which cleaved the fatty acids at the sn-1 and sn-2 positions of phosphatidylcholine. However, it did not show lysophospholipase, sphingomyelinase, and phospholipase C activities. The rPhlA showed maximum activity at temperature of about 40 °C and pH around 8–9. Some divalent cations could affect the activity of PhlA. The addition of Co 2+ increased the activity, whereas Mg 2+ and Zn 2+ did not enhance the enzyme activity. The rPhlA could lyse the erythrocytes obtained from the fish such as rainbow trout and tilapia. A significant cytotoxic activity on a fish cell line, CHSE-214, was observed after 24 h exposure to 40 μ g rPhlA protein.
ISSN:0006-291X
1090-2104
DOI:10.1016/S0006-291X(02)02434-8