Purification and properties of a monoacylglycerol lipase in human erythrocytes.
A membrane-bound monoacylglycerol lipase (MAGL) activity, previously demonstrated in intact human erythrocytes [Boyer, Somma, Vérine, L'Hôte, Finidori, Merger and Arnaud (1981) J. Clin. Endocrinol. Metab. 53, 143-148], has now been purified to apparent homogeneity by a five-step procedure involving solubilization in CHAPS and sequential chromatographies on Sephacryl S-400, DEAE-Trisacryl, Zn(2+)-chelating Sepharose and Superose 12 columns. The purified protein has a molecular mass of 68 +/- 2 kDa, as determined by SDS/PAGE and gel filtration, suggesting that the enzyme behaves as a monomer. The concentration-dependence of MAGL activity with monooleoylglycerol, the preferred substrate showed kinetics typical of an interfacial lipolytic enzyme displaying optimal activity on emulsified substrate particles; apparent Km values were 0.27 mM and 0.49 mM for the sn-1(3)- and sn-2-isomers respectively. MAGL had no, or negligible, activity towards tri-oleoylglycerol, di-oleoylglycerol, oleoylcholesterol, oleoyl-CoA and phosphatidylcholine; it was inhibited by di-isopropylfluorophosphate, PMSF and diethyl p-nitrophenyl phosphate, suggesting that MAGL is a serine hydrolase. MAGL activity was not modified by bile salt or apolipoprotein C-II, whereas a dose-dependent inhibition was observed with apolipoprotein A-I.
Somma-Delpéro
C
C
INSERM U. 260, Faculté de Médecine, Marseille, France.
Valette
A
A
Lepetit-Thévenin
J
J
Nobili
O
O
Boyer
J
J
Vérine
A
A
D016632:Apolipoprotein A-I / Q000494:pharmacology; D002793:Cholic Acids / Q000494:pharmacology; D002846:Chromatography, Affinity; D002850:Chromatography, Gel; D004591:Electrophoresis, Polyacrylamide Gel; D004791:Enzyme Inhibitors / Q000494:pharmacology; D004910:Erythrocyte Membrane / Q000201:enzymology*; D006801:Humans; D007700:Kinetics; D008994:Monoacylglycerol Lipases / Q000097:blood* / Q000737:chemistry / Q000302:isolation & purification*; D010261:Paraoxon / Q000494:pharmacology; D013379:Substrate Specificity
D016632:Apolipoprotein A-I; D002793:Cholic Acids; D004791:Enzyme Inhibitors; D008994:Monoacylglycerol Lipases; D010261:Paraoxon; C028213:3-((3-cholamidopropyl)dimethylammonium)-1-propanesulfonate