Purification and characterization of diacylglycerol lipase from human platelets

Diacylglycerol lipase (DGL) was solubilized from human platelet microsomes with heptyl-beta-D-thioglucoside, and purified to homogeneity on SDS-PAGE u sing a combination of chromatographic and electrophoretic methods. The mole cular mass of the purified DGL was estimated to be 33 kDa. Its apparent pi was pH 6.0, as determined by Immobiline isoelectrofocusing. The enzymatic a ctivity of the partially purified DGL was investigated in the presence of a variety of inhibitors and reagents, as well as its pH and calcium dependen ce, Thiol reagents such as p-chloromercurubenzoic acid (pCMB), N-ethylmalei mide (NEM), and HgCl2, inhibited the activity, while dithiothreitol (DTT) a nd reduced glutathione (GSH) enhanced it. In addition, the enzymatic activi ty was inhibited by two serine blockers, phenylmethylsulfonyl fluoride (PMS F) and diisopropyl fluorophosphate (DFP), and by a histidine modifying reag ent, p-bromophenacyl bromide (pBPB). These results suggest that cysteine, s erine and histidine residues are required for the enzymatic activity of DGL , DGL was optimally active in the pH range of 7-8 and its activity did not change significantly in the presence of various calcium concentrations, eve n in the presence of 2 mM EGTA. This indicates that DG;L can hydrolyze subs trates with a basal cytosolic free Ca2+ level in the physiological pH range . A DGL inhibitor, RHC-80267, inhibited DGL activity in a dose-dependent ma nner with an IC50 (the concentration required for 50% inhibition) of about 5 mu M, Unexpectedly, several phospholipase Az (PLA,) inhibitors were poten t inhibitors of DCL activity (IC50 < 5 mu M), suggesting that the catalytic mechanisms of DGL and PLA(2) may be similar. Finally, we show that DGL act ivity was inhibited by 2-monoacylglycerols (2-MGs), the reaction products o f this enzyme, Among the three 2-MGs tested (2-arachidonoyl glycerol, a-ste aroyl glycerol, and a-oleoyl glycerol), 2-arachidonoyl glycerol was the mos t potent inhibitor.