Differential effects of manoalide on secreted and intracellular phospholipases

Abstract

Manoalide, a novel nonsteroidal sesterterpenoid, is a potent inhibitor of phospholipase A 2 isolated from bee and cobra venoms. This report compares the inhibition by manoalide of phospholipase A 2 in crude cytosol fractions from four mammalian tissues with that of four purified extracellular phospholipase A 2's. Phospholipase A 2 isolated from bee venom ( Apis mellifera) was the most sensitive to inactivation by manoalide ( ic 50 ⋍ 0.12 μ M ). Extracellular phospholipase A 2 from rattlesnake and cobra venom was intermediate in sensitivity to manoalide ( ic 50 values of 0.7 and 1.9 μM respectively). Porcine pancreatic phospholipase A 2 was relatively resistant to inactivation by manoalide ( ic 50 ⋍ 30 μ M ). The phospholipase A 2 assayed in crude cytosol fractions from four mammalian tissues exhibited ic 50 values of 30 μM or greater. Cytosolic proteins as well as bovine serum albumin and poly-L-lysine ( Mr = 57,000) protected purified bee venom phospholipase A 2 from inactivation by manoalide. In contrast, amino acids such as lysine and alanine failed to protect the purified enzyme from inactivation. Proteins and certain amino acids, such as lysine, formed a chromogenic product when incubated with manoalide. These data suggest that lysine is capable of reacting with manoalide, but only when it is present in macromolecules is it capable of protecting phospholipase A 2 from inactivation by manoalide. Because cellular proteins protect PLA 2 from inactivation by manoalide, high concentrations of manoalide must be applied topically to produce statistically significant inactivation of intracellular phospholipase A 2. Finally, a chemical model is presented which explains the formation of a chromogenic product when manoalide is incubated with proteins and amino acids.