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 invol...

Full description

Saved in:
Bibliographic Details
Published in:Biochemical journal 1995-12, Vol.312 ( Pt 2) (2), p.519-525
Main Authors: Somma-Delpéro, C, Valette, A, Lepetit-Thévenin, J, Nobili, O, Boyer, J, Vérine, A
Format: Article
Language:English
Subjects:
Apolipoprotein A-I - pharmacology
Cholic Acids - pharmacology
Chromatography, Affinity
Chromatography, Gel
Electrophoresis, Polyacrylamide Gel
Enzyme Inhibitors - pharmacology
Erythrocyte Membrane - enzymology
Humans
Kinetics
Monoacylglycerol Lipases - blood
Monoacylglycerol Lipases - chemistry
Monoacylglycerol Lipases - isolation & purification
Paraoxon - pharmacology
Substrate Specificity
Citations: Items that cite this one
Online Access:Get full text
Tags: Add Tag
No Tags, Be the first to tag this record!
Description
Summary: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.
ISSN:0264-6021
1470-8728
DOI:10.1042/bj3120519