Suicide inhibition of canine myocardial cytosolic calcium-independent phospholipase A₂: Mechanism-based discrimination between calcium-dependent and -independent phospholipases A₂

The majority of phospholipase A₂ activity in myocardium is calcium-independent and selective for hydrolysis of plasmalogen substrate (Wolf, R. A., and Gross, R. W. (1985) J. Biol. Chem. 260, 7295-7303; Hazen, S. L., Stuppy, R. J., and Gross, R. W. (1990) J. Biol. Chem. 265, 10622-10630). Accordingly, identification of an inhibitor which selectively targets calcium-independent phospholipases A₂ would facilitate elucidation of the biologic significance of this class of intracellular phospholipases. We now report that the haloenol lactone, (E)-6-(bfomomethylene)tetrahydro-3-(1-naphthalenyl)-2H-pyran-2-one (Compound 1), is a potent, irreversible, mechanism-based inhibitor of myocardial calcium-independent phospholipase A₂ which is > 1000-fold specific for inhibition of myocardial calcium-independent phospholipase A₂ in comparisons with multiple calcium-dependent phospholipases A₂. Mechanism-based inhibition of myocardial cytosolic calcium-independent phospholipase A₂ by Compound 1 was established by demonstrating: 1) time-dependent irreversible inactivation; 2) covalent binding of [³H]Compound 1 to the purified phospholipase A₂; 3) ablation of covalent binding of [³H]Compound 1 after chemical inactivation of phospholipase A₂ enzymic activity; 4) identical inhibition of myocardial phospholipase A₂ by Compound 1 in the absence or presence of nucleophilic scavengers; 5) Compound 1 is a substrate for myocardial calcium-independent phospholipase A₂ resulting in the generation of the electrophilic α-bromomethyl ketone; 6) phospholipase A₂ inhibition requires the in situ generation of the reactive electrophile (i.e. neither the α-bromomethyl ketone nor the diproteoenol lactone analog are inhibitory); and 7) concomitant attenuation of the inhibitory potency and the extent of covalent adduct formation in the presence of saturating substrate. Collectively, these results demonstrate that the haloenol lactone, Compound 1, is a substrate for, covalently binds to, and irreversibly inhibits canine myocardial cytosolic calcium-independent phospholipase A₂.