Activation of phospholipase A(2) by long chain fatty acyl groups involves a novel unstable linkage

The acidic isoform of phospholipase A(2) from Naja mossambica mossambica wa s activated by treatment with a molar equivalent of oleoyl imidazolide. Mod ification of the protein was accompanied by 50% quenching of tryptophan flu orescence and a significant red shift. The H-3(9,10) labeled oleoyl residue was co-eluted with the enzyme during gel filtration in the presence of 20% 1-propanol or excess albumin, both of which remove free oleic acid from th e enzyme. In contrast, the adduct was labile as to electrophoresis on SDS-P AGE and acid or alkali urea PAGE. The formation of a covalently linked addu ct was demonstrated by electrospray mass spectrometry in the presence of 2% formic acid. No such adduct was formed by the phospholipase A(2) isoform f rom Naja naja atra, which differs in sequence from the N. mossambica mossam bica isoform by seven residues including 2 histidine residues and 1 lysine residue. We conclude that oleoyl imidazolide activates the N. mossambica mo ssambica enzyme by forming an acyl adduct which is unstable as to protein d enaturation. The magnitude of tryptophan fluorescence quenching indicates t hat the site of acylation lies in the sequence WWHF.