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-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 degrees 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 24h exposure to 40 microg rPhlA protein.
Lee
Jong-Hee
JH
Department of Biotechnology and Bioengineering, Pukyong National University, Busan 608-737, Republic of Korea.
Ahn
Sun-Hee
SH
Kim
Sun-Hoi
SH
Choi
Yoon-Hyeok
YH
Park
Kee-Jai
KJ
Kong
In-Soo
IS
D000818:Animals; D002460:Cell Line; D004926:Escherichia coli / Q000235:genetics; D005399:Fishes; D006460:Hemolysin Proteins / Q000235:genetics / Q000378:metabolism / Q000494:pharmacology; D006863:Hydrogen-Ion Concentration; D008670:Metals / Q000494:pharmacology; D010741:Phospholipases A / Q000235:genetics / Q000378:metabolism* / Q000633:toxicity*; D010743:Phospholipids / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000302:isolation & purification; D013379:Substrate Specificity; D013696:Temperature; D014733:Vibrio / Q000201:enzymology*
D006460:Hemolysin Proteins; D008670:Metals; D010743:Phospholipids; D011993:Recombinant Fusion Proteins; D010741:Phospholipases A