Kinase experiment:

MAFP is dissolved and diluted in DMSO. To investigate the reversibility of iPLA 2 inhibition by MAFP, the P388D1 iPLA 2 is first concentrated approximately 10-fold using a Centricon-10 concentrator from Amicon. The concentrated iPLA 2 (20 μL) is then preincubated with either 80 μM MAFP in DMSO or DMSO alone (2 μL) for 5 min at 40℃. A 2 μL aliquot is removed and subsequently diluted 1500-fold into 3 mL of assay mixture containing 100 μM DPPC (200000 cpm per 50 μL assay mixture), 400 μM Triton X-100, 100 mM Hepes (pH 7.5), 5 mM EDTA, 1 mM DTT and 0.8 mM ATP. At the indicated time points, a 50 μL aliquot is removed and the remaining enzyme activity is quantified[1].

Cell experiment:

Inhibition of anandamide amidase in cell culture is measured using approximately 1x106 Nl8TG2 intact neuroblastoma cells. Experimental cells are preincubated for 20 min in 1.5 mL medium, consisting of Fl2/DMEM with penicillin, streptomycin, gentamicin, 10% bovine calf serum, plus MAFP (1, 5, 10, 20 nM). Control cells contained no inhibitor. Arachidonoyl is then added and the incubation continued for I hr. The amount of [3H]anandamide in the cells is quantified by liquid scintillation counting of the silica scraped from the appropriate areas of the TLC plate identified by exposure to X-ray film[2].

Methyl arachidonyl fluorophosphonate (MAFP) is a selective, active-site directed, irreversible inhibitor of cPLA2 and iPLA2.[1],[2],[3] It inhibits A23187-induced arachidonic acid release from human platelets with an IC50 value of 0.6 μM.1 The IC50 value for inhibition of iPLA2 from P388D1 cells is 0.5 μM.2 MAFP is a potent inhibitor of fatty acid amide hydrolase, exhibiting an IC50 value of 2.5 nM. MAFP also binds to the CB1 receptor in rat brain membrane preparations. The IC50 values for MAFP and arachidonyl ethanolamide in this assay are 20 and 40 nM, respectively.[4]

Reference:
[1]. Huang, Z., Liu, S., Street, I., et al. Methyl arachidonyl fluorophosphonate, a potent irreversible cPLA2 inhibitor, blocks the mobilization of arachidonic acid in human platelets and neutrophils. Mediators of Inflammation 3, 307-308 (1994).
[2]. Lio, Y.C., Reynolds, L.J., Balsinde, J., et al. Irreversible inhibition of Ca2+-independent phospholipase A2 by methyl arachidonyl fluorophosphonate. Biochimica et Biophysica Acta 1302, 55-60 (1996).
[3]. Balsinde, J., and Dennis, E.A. Distinct roles in signal transduction for each of the phospholipase A2 enzymes present in P388D1 macrophages. The Journal of Biological Chemisty 271, 6758-6765 (1996).
[4]. Deutsch, D.G., Omeir, R., Arreaza, G., et al. Methyl arachidonyl fluorophosphonate: A potent irreversible inhibitor of anandamide amidase. Biochemical Pharmacology 53, 255-260 (1997).