Ostrich pancreatic phospholipase A(2): purification and biochemical characterization.
Ostrich pancreatic phospholipase A(2) (OPLA(2)) was purified from delipidated pancreases. Pure protein was obtained after heat treatment (70 degrees C), precipitation by ammonium sulphate and ethanol, respectively followed by sequential column chromatography on MonoQ Sepharose and size exclusion HPLC column. Purified OPLA(2), which is not a glycosylated protein, was found to be monomeric protein with a molecular mass of 13773.93 Da. A specific activity of 840U/mg for purified OPLA(2) was measured at optimal conditions (pH 8.2 and 37 degrees C) in the presence of 4 mM NaTDC and 10 mM CaCl(2) using PC as substrate. This enzyme was also found to be able to hydrolyze, at low surface pressure, 1,2-dilauroyl-sn-glycero-3 phosphocholine (di C(12)-PC) monolayers. Maximal activity was measured at 5-8 mNm(-1). The sequence of the first 22 amino-acid residues at the N-terminal extremity of purified bird PLA(2) was determined by automatic Edman degradation and showed a high sequence homology with known mammal pancreatic secreted phospholipases A(2).
Ben Bacha
Abir
A
Laboratoire de Biochimie et de Génie Enzymatique des Lipases, ENIS route de Soukra, 3038, Sfax, Tunisia.
Gargouri
Youssef
Y
Bezzine
Sofiane
S
Mosbah
Habib
H
Mejdoub
Hafedh
H
D000645:Ammonium Sulfate / Q000737:chemistry; D000818:Animals; D001717:Birds / Q000378:metabolism; D011232:Chemical Precipitation; D002851:Chromatography, High Pressure Liquid / Q000379:methods; D002852:Chromatography, Ion Exchange / Q000379:methods; D004789:Enzyme Activation; D008970:Molecular Weight; D010179:Pancreas / Q000201:enzymology*; D010713:Phosphatidylcholines / Q000378:metabolism; D054467:Phospholipases A2 / Q000302:isolation & purification* / Q000378:metabolism*; D010802:Phylogeny; D012117:Resins, Synthetic / Q000737:chemistry; D016415:Sequence Alignment; D017386:Sequence Homology, Amino Acid; D013045:Species Specificity; D020310:Struthioniformes; D013696:Temperature
D010713:Phosphatidylcholines; D012117:Resins, Synthetic; C042855:Mono Q; D054467:Phospholipases A2; D000645:Ammonium Sulfate