Human myocardial cytosolic Ca²⁺-independent phospholipase A₂ is modulated by ATP: Concordant ATP-induced alterations in enzyme kinetics and mechanism-based inhibition

Although phospholipases A₂ (PLsA₂) have been implicated as enzymic mediators of electrophysiological dysfunction during myocardial ischaemia and reperfusion in man, no information on the regulation of human myocardial PLsA₂ is available. We now report that human myocardial cytosolic Ca²⁺-independent PLA₂ is modulated by ATP through mechanisms which are independent of protein phosphorylation, since: (1) ATP and its non-hydrolysable analogues reversibly augment the initial rate of crude (but not purified) human myocardial cytosolic PLA₂ activity 3-4-fold; (2) ATP and its non-hydrolysable analogues dramatically attenuate the rate of thermal denaturation of human myocardial cytosolic PLA₂ activity; (3) ATP and its non-hydrolysable analogues alter the sensitivity of human myocardial cytosolic PLA₂ to mechanism-based inhibition by (E)-6-(bromomethylene)tetrahydro-3-(1-naphthalenyl)-2H- pyran-2-one; (4) adenine nucleotide triphosphates are more potent than guanine nucleotide triphosphates in activating and stabilizing human myocardial cytosolic PLA₂; (5) adenosine 5'-[βγ-methylene]triphosphate is more potent than its guanosine counterpart in protecting an essential thiol residue(s) in human myocardial cytosolic PLA₂ from covalent modification by 5,5'-dithiobis-(2-nitrobenzoic acid); and (6) ATP-dependent activation and stabilization of human myocardial cytosolic PLA₂ are mediated by a cytosolic protein(s) which can be functionally reconstituted with purified human myocardial cytosolic PLA₂ catalytic polypeptide. Collectively these results demonstrate that multiple physical and kinetic properties of human myocardial cytosolic Ca²⁺-independent PLA₂ are dramatically influenced by dynamic interactions with ATP.