摘要：

The serine hydrolase monoacylglycerol lipase (MGL) modulates endocannabinoid signaling by inactivating 2-arachidonoylglycerol (2-AG), the main endogenous agonist for central CB1 and peripheral CB2 cannabinoid receptors. To characterize this key endocannabinoid enzyme by mass spectrometry-based proteomics, we first overexpressed recombinant hexa-histidine-tagged human MGL (hMGL) in and purified it in a single chromatographic step with high yield (≈30 mg/L). With 2-AG as substrate, hMGL displayed an apparent of 25 mol/(g min) and of 19.7 M, an affinity for 2-AG similar to that of native rat-brain MGL (rMGL) (= 33.6 M). hMGL also demonstrated a comparable affinity (≈ 8–9 M) for the novel fluorogenic substrate, arachidonoyl, 7-hydroxy-6-methoxy-4-methylcoumarin ester (AHMMCE), in a sensitive, high-throughput fluorometric MGL assay. Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) unequivocably demonstrated the mass (34126 Da) and purity of this hMGL preparation. After in-solution tryptic digestion, hMGL full proteomic characterization was carried out, which showed (1) an absence of intramolecular disulfide bridges in the functional, recombinant enzyme and (2) the post-translational removal of the enzyme's N-terminal methionine. Availability of sufficient quantities of pure, well-characterized hMGL will enable further molecular and structural profiling of this key endocannabinoid-system enzyme.

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