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 using a combination of chromatographic and electrophoretic methods. The molecular mass of the purified DGL was estimated to be 33 kDa. Its apparent pI was pH 6.0, as determined by Immobiline isoelectro-focusing. The enzymatic activity of the partially purified DGL was investigated in the presence of a variety of inhibitors and reagents, as well as its pH and calcium dependence. Thiol reagents such as p-chloromercurubenzoic acid (pCMB), N-ethylmaleimide (NEM), and HgCl2 inhibited the activity, while dithiothreitol (DTT) and reduced glutathione (GSH) enhanced it. In addition, the enzymatic activity was inhibited by two serine blockers, phenylmethylsulfonyl fluoride (PMSF) and diisopropyl fluorophosphate (DFP), and by a histidine modifying reagent, p-bromophenacyl bromide (pBPB). These results suggest that cysteine, serine 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, even in the presence of 2 mM EGTA. This indicates that DGL can hydrolyze substrates with a basal cytosolic free Ca2+ level in the physiological pH range. A DGL inhibitor, RHC-80267, inhibited DGL activity in a dose-dependent manner with an IC50 (the concentration required for 50% inhibition) of about 5 microM. Unexpectedly, several phospholipase A2 (PLA2) inhibitors were potent inhibitors of DGL activity (IC50<5 microM), suggesting that the catalytic mechanisms of DGL and PLA2 may be similar. Finally, we show that DGL activity was inhibited by 2-monoacylglycerols (2-MGs), the reaction products of this enzyme. Among the three 2-MGs tested (2-arachidonoyl glycerol, 2-stearoyl glycerol, and 2-oleoyl glycerol), 2-arachidonoyl glycerol was the most potent inhibitor.
Moriyama
T
T
Research Institute for Food Science, Kyoto University, Uji, Kyoto 611-0011, Japan. moriyama@soya.food.kyoto-u.ac.jp
Urade
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R
Kito
M
M
D001792:Blood Platelets / Q000201:enzymology*; D020134:Catalytic Domain; D003902:Detergents; D004791:Enzyme Inhibitors / Q000494:pharmacology; D006801:Humans; D066298:In Vitro Techniques; D007526:Isoelectric Point; D008071:Lipoprotein Lipase / Q000097:blood / Q000737:chemistry / Q000302:isolation & purification*; D008861:Microsomes / Q000201:enzymology; D008970:Molecular Weight; D012995:Solubility
D003902:Detergents; D004791:Enzyme Inhibitors; D008071:Lipoprotein Lipase