title: Activation of phospholipase A(2) by long chain fatty acyl groups involves a novel unstable linkage.
abstract: The acidic isoform of phospholipase A(2) from Naja mossambica mossambica was activated by treatment with a molar equivalent of oleoyl imidazolide. Modification of the protein was accompanied by 50% quenching of tryptophan fluorescence and a significant red shift. The (3)H(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 the enzyme. In contrast, the adduct was labile as to electrophoresis on SDS-PAGE and acid or alkali urea PAGE. The formation of a covalently linked adduct was demonstrated by electrospray mass spectrometry in the presence of 2% formic acid. No such adduct was formed by the phospholipase A(2) isoform from Naja naja atra, which differs in sequence from the N. mossambica mossambica isoform by seven residues including 2 histidine residues and 1 lysine residue. We conclude that oleoyl imidazolide activates the N. mossambica mossambica enzyme by forming an acyl adduct which is unstable as to protein denaturation. The magnitude of tryptophan fluorescence quenching indicates that the site of acylation lies in the sequence WWHF.
authors.affiliation: Division of Infection and Immunity, Institute of Biomedical and Life Sciences, Joseph Black Building, University of Glasgow, Glasgow, G12 8QQ, UK. bmbta@leeds.ac.uk
authors.forename: T
authors.initials: T
authors.lastname: Ahmed
authors.forename: S M
authors.initials: SM
authors.lastname: Kelly
authors.forename: N C
authors.initials: NC
authors.lastname: Price
authors.forename: A J
authors.initials: AJ
authors.lastname: Lawrence
mesh_terms: D000215:Acylation; D000818:Animals; D002850:Chromatography, Gel; D004546:Elapid Venoms / Q000201:enzymology*; D017815:Elapidae; D004586:Electrophoresis; D004789:Enzyme Activation; D005227:Fatty Acids / Q000378:metabolism*; D013058:Mass Spectrometry; D010741:Phospholipases A / Q000378:metabolism*; D011499:Protein Processing, Post-Translational; D013050:Spectrometry, Fluorescence
chemical_list: D004546:Elapid Venoms; D005227:Fatty Acids; D010741:Phospholipases A