WAY-195725

The cytosolic (group IV) phospholipase A(2) (cPLA(2)s) family contains six members. We have prepared recombinant proteins for human α, mouse β, human γ, human δ, human ε, and mouse ζ cPLA(2)s and have studied their interfacial kinetic and binding properties in vitro. Mouse cPLA(2)β action on phosphatidylcholine vesicles is activated by anionic phosphoinositides and cardiolipin but displays a requirement for Ca(2+) only in the presence of cardiolipin. This activation pattern is explained by the effects of anionic phospholipids and Ca(2+) on the interfacial binding of mouse cPLA(2)β and its C2 domain to vesicles. Ca(2+)-dependent binding of mouse cPLA(2)β to cardiolipin-containing vesicles requires a patch of basic residues near the Ca(2+)-binding surface loops of the C2 domain, but binding to phosphoinositide-containing vesicles does not depend on any specific cluster of basic residues. Human cPLA(2)δ also displays Ca(2+)- and cardiolipin-enhanced interfacial binding and activity. The lysophospholipase, phospholipase A(1), and phospholipase A(2) activities of the full set of mammalian cPLA(2)s were quantified. The relative level of these activities is very different among the isoforms, and human cPLA(2)δ stands out as having relatively high phospholipase A(1) activity. We also tested the susceptibility of all cPLA(2) family members to a panel of previously reported inhibitors of human cPLA(2)α and analogs of these compounds. This led to the discovery of a potent and selective inhibitor of mouse cPLA(2)β. These in vitro studies help determine the regulation and function of the cPLA(2) family members.

Cytosolic phospholipase A(2) alpha (cPLA(2)alpha, type IVA phospholipase) acts at the membrane surface to release free arachidonic acid, which is metabolized into inflammatory mediators, including leukotrienes and prostaglandins. Thus, specific cPLA(2)alpha inhibitors are predicted to have antiinflammatory properties. However, a key criterion in the identification and development of such inhibitors is to distinguish between compounds that bind stoichiometrically to cPLA(2)alpha and nonspecific membrane perturbants. In the current study, we developed a method employing isothermal titration calorimetry (ITC) to characterize the binding of several distinct classes of cPLA(2)alpha inhibitors. Thermodynamic parameters and the binding constants were obtained following titration of the inhibitor to the protein at 30 degrees C and pH 7.4. The compounds tested bound cPLA(2)alpha with a 1:1 stoichiometry, and the dissociation constant K(d) of the inhibitors calculated from the ITC experiments correlated well with the IC(50) values obtained from enzymatic assays. Interestingly, binding was observed only in the presence of a micellar surface, even for soluble compounds. The site of binding of these inhibitors within cPLA(2)alpha was analyzed by testing for binding in the presence of methyl arachidonyl fluorophosphonate (MAFP), an irreversible active site inhibitor of cPLA(2)alpha. Lack of binding of inhibitors in the presence of MAFP suggested that the compounds tested bound specifically at or near the active site of the protein. Furthermore, the effect of various detergents on the binding of certain inhibitors to cPLA(2)alpha was also tested. The results are discussed with reference to thermodynamic parameters such as changes in enthalpy (DeltaH), entropy (DeltaS), and free energy (DeltaG). The data obtained from these studies provide not only structure-activity relationships for compounds but also important information regarding mechanism of binding. This is the first example of ITC used for studying inhibitors of enzymes with interfacial kinetics.