Contribution of bee venom phospholipase A2 contamination in melittin fractions to presumed activation of tissue phospholipase A2.

ABSTRACT

Melittin from bee venom has been suggested to activate tissue phospholipase A2 (PLA2) activity, and subsequently has been used as a specific PLA2 probe. The melittin in most cases was obtained commercially and used without further purification or treatment. To test the hypothesis that commercially obtained melittin specifically activates tissue PLA2, we radiolabeled the lipids of immortalized epithelial cells by incubating the cells for 22 hr with 14C-linoleic acid. The cells were then incubated with 2 microM melittin, 2nM bee venom PLA2, 2 microM melittin treated with p-bromophenacyl bromide (p-BPB) or PLA2 plus p-BPB-treated melittin. Lipids were extracted and separated by thin-layer chromatography. The radioactivity in each lipid fraction was then quantitated. The melittin-stimulated PLA2 activity observed in cells was primarily associated with phosphatidylcholine. Fatty acid release was decreased by 75% when the melittin fraction was pretreated with p-BPB to reduce contaminating venom PLA2 activity. Adding PLA2 to the p-BPB-treated melittin at an amount about equal to the original contamination (0.1%) resulted in the same PLA2 activity in cell as observed with the untreated melittin fraction. These findings suggest that bee venom PLA2 contamination, even at very low levels, can account for approximately 75% of the PLA2 activity in cells treated with commercial melittin fractions.