Purification of rat liver mitochondrial phospholipase A2

Rat liver mitochondrial phospholipase A2 was purified to near homogeneity by a combination of gel-filtration, hydroxyapatite and Matrex gel Blue A column chromatography. The absolute positional specificity of the enzyme for acylester bonds at the sn-2-position was established in experiments with 1-[9,10-3H2]palmitoyl-2-[1-14C]linoleoylphosphatidylethanolamine. Molecular weight estimations revealed Mr values of 15000 by SDS-polyacrylamide gel electrophoresis and of 9700 gel by gel-filtration over Ultrogel AcA 54 columns. The enzyme is unaffected by diisopropylfluorophosphate and thiol reagents such as 5,5'-dithiobis(2-nitrobenzoic acid), N-ethylmaleimide and iodoacetamide, but is completely inhibited by the alkylating reagent p-bromophenacylbromide.