Purification and characterization of a cytosolic phospholipase A2 from rat liver.
A cytosolic phospholipase A2 (PLA2) was purified 640-fold from rat liver by sequential anion-exchange chromatography, Ca2+-precipitation/KCl-solubilization, gel filtration chromatography, and affinity chromatography. A single peak of PLA2 activity was eluted at an apparent molecular mass of 197 kDa from a Superdex 200HR gel filtration column. In the presence of Ca2+, the purified enzyme catalyzed the hydrolysis of 81.8 nmol of phosphatidylethanolamine per hour per mg of protein. The apparent Km was 1.83 nM. The enzyme was inhibited by arachidonyl trifluoromethyl ketone (AACOCF3), an inhibitor of cPLA2. However, it was not inhibited by bromoenol lactone (BEL), an inhibitor of iPLA2, and p-bromophenacyl bromide (p-BPB), an inhibitor of sPLA2. These data suggest that the purified enzyme is a novel Ca2+-dependent cytosolic PLA2.
Lee
Dongkoo
D
Department of Biological Sciences, Institute for Basic Sciences, Sungkyunkwan University, Suwon 440-746, Korea.
Won
Jong Hak
JH
Auh
Chung-Kyoon
CK
Park
Young Min
YM
D000818:Animals; D002118:Calcium / Q000378:metabolism; D003600:Cytosol / Q000201:enzymology*; D004591:Electrophoresis, Polyacrylamide Gel; D008099:Liver / Q000201:enzymology*; D008970:Molecular Weight; D010741:Phospholipases A / Q000302:isolation & purification* / Q000378:metabolism; D054467:Phospholipases A2; D051381:Rats
D010741:Phospholipases A; D054467:Phospholipases A2; D002118:Calcium