Inactivation of human synovial fluid phospholipase a₂ by the marine natural product, manoalide

Abstract

The marine natural product, manoalide (MLD), was investigated to determine if this drug inhibited purified human synovial fluid phospholipase A₂ (HSF-PLA₂). Utilizing classical Michaelis-Menten kinetics, apparent K_m and V_max, values for HSF-PLA₂ of 1.34mM and 0.47μmol [³H]palmitic acid released/min/mg protein were obtained using dipalmitoylphosphatidylcholine (DPPC) as the substrate, and 38.0 μM and 18.8 μmol [³H]arachidonic acid released/min/mg protein with Escherichia coli as a natural substrate. These kinetic parameters were utilized subsequently to evaluate the inhibitory effects of manoalide on HSF-PLA₂. Inhibition of HSF-PLA₂ by MLD was concentration and time dependent with ic₅₀ values of 0.2 and 0.02μM for DPPC and E. coli respectively. Dialysis studies and examination of DPPC or E. coli hydrolysis versus enzyme concentration indicate that MLD is an irreversible inhibitor of HSF-PLA₂. Substrate specificity was also examined in the absence and presence of MLD using dipalmitoylphosphatidylethanolamine (DPPE) as a substrate. MLD inhibited the hydrolysis of DPPE (>90% inhibition at μM), and preliminary results indicate that DPPC was more readily hydrolyzed than DPPE under the substrate conditions of the assay. While the cellular source of secreted HSF-PLA₂ is unknown, these studies indicate that MLD can inactivate secreted phospholipase A₂ isolated from patients with inflammatory joint disease.