Characterization of the sulfhydryl-sensitive site in the enzyme responsible for hydrolysis of 2-arachidonoyl-glycerol in rat cerebellar membranes.
We have previously reported that the endocannabinoid, 2-arachidonoyl-glycerol (2-AG), is hydrolyzed in rat cerebellar membranes by monoglyceride lipase (MGL)-like enzymatic activity. The present study shows that, like MGL, 2-AG-degrading enzymatic activity is sensitive to inhibition by sulfhydryl-specific reagents. Inhibition studies of this enzymatic activity by N-ethylmaleimide analogs revealed that analogs with bulky hydrophobic N-substitution were more potent inhibitors than hydrophilic or less bulky agents. Interestingly, the substrate analog N-arachidonylmaleimide was found to be the most potent inhibitor. A comparison model of MGL was constructed to get a view on the cysteine residues located near the binding site. These findings support our previous conclusion that the 2-AG-degrading enzymatic activity in rat cerebellar membranes corresponds to MGL or MGL-like enzyme and should facilitate further efforts to develop potent and more selective MGL inhibitors.
Department of Pharmaceutical Chemistry, P.O. Box 1627, FIN-70211 Kuopio, Finland. susanna.saario@uku.fi
Susanna M
SM
Saario
Outi M H
OM
Salo
Tapio
T
Nevalainen
Antti
A
Poso
Jarmo T
JT
Laitinen
Tomi
T
Järvinen
Riku
R
Niemi
D000595:Amino Acid Sequence; D000818:Animals; D001095:Arachidonic Acids / Q000378:metabolism*; D001665:Binding Sites; D002384:Catalysis; D002462:Cell Membrane / Q000201:enzymology / Q000378:metabolism*; D002531:Cerebellum / Q000166:cytology* / Q000201:enzymology*; D002711:Chloride Peroxidase / Q000737:chemistry / Q000378:metabolism; D003545:Cysteine / Q000378:metabolism; D063388:Endocannabinoids; D004791:Enzyme Inhibitors / Q000737:chemistry / Q000494:pharmacology; D005989:Glycerides / Q000378:metabolism*; D006868:Hydrolysis; D020128:Inhibitory Concentration 50; D008297:Male; D008958:Models, Molecular; D008969:Molecular Sequence Data; D015394:Molecular Structure; D008994:Monoacylglycerol Lipases / Q000737:chemistry / Q000378:metabolism; D017434:Protein Structure, Tertiary; D051381:Rats; D017208:Rats, Wistar; D016415:Sequence Alignment; D048170:Streptomyces lividans / Q000201:enzymology; D013329:Structure-Activity Relationship; D013438:Sulfhydryl Compounds / Q000737:chemistry*
D001095:Arachidonic Acids; D063388:Endocannabinoids; D004791:Enzyme Inhibitors; D005989:Glycerides; D013438:Sulfhydryl Compounds; C094503:glyceryl 2-arachidonate; D002711:Chloride Peroxidase; D008994:Monoacylglycerol Lipases; D003545:Cysteine