High-Performance Liquid Chromatographic Determination of Anandamide Amidase Activity in Rat Brain Microsomes

Lang, Wensheng; Qin, Ce; Hill, Winfield; Lin, Sonyuan; Khanolkar, Atmaram D.; Makriyannis, A.

doi:10.1006/abio.1996.0247

Cited by 40 publications

(31 citation statements)

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“…Crude rat-brain MGL (rMGL) was prepared as described. 18 Kits for protein determination by the Bradford dyebinding method were from Bio-Rad Laboratories (Hercules, CA).…”

Section: Experimental Section Materialsmentioning

confidence: 99%

“…In the first, hydrolysis of 2-AG to AA was quantified by HPLC. 18 Briefly, 2-AG at varying concentrations from 12.7 to 400 μM and hMGL (0.5-2.0 ng) in TME buffer (150 μL) were incubated at 37 °C. Reaction samples (50 μL) were taken immediately at the start of the incubation and after 20 min, diluted 1:4 with acetonitrile, and centrifuged at 20 000g for 5 min at room temperature.…”

Section: Mgl Assaymentioning

confidence: 99%

“…A 10-μL aliquot of supernatant was injected onto the HPLC, with chromatographic conditions previously detailed. 18 In an 8-min HPLC run, 2-AG eluted at 3.0 min, and AA at 6.0 min, allowing the reaction to be followed by either substrate (2-AG) turnover or product (AA) formation. Analytes were quantified with external standards.…”

Section: Mgl Assaymentioning

confidence: 99%

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Full Mass Spectrometric Characterization of Human Monoacylglycerol Lipase Generated by Large-Scale Expression and Single-Step Purification

et al. 2008

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The serine hydrolase monoacylglycerol lipase (MGL) modulates endocannabinoid signaling in vivo 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 Escherichia coli and purified it in a single chromatographic step with high yield (≈30 mg/L). With 2-AG as substrate, hMGL displayed an apparent V max of 25 μmol/ (μg min) and K m of 19.7 μM, an affinity for 2-AG similar to that of native rat-brain MGL (rMGL) (K m = 33.6 μM). hMGL also demonstrated a comparable affinity (K m ≈8-9 μM) 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 (34 126 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, wellcharacterized hMGL will enable further molecular and structural profiling of this key endocannabinoid-system enzyme.

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“…Crude rat-brain MGL (rMGL) was prepared as described. 18 Kits for protein determination by the Bradford dyebinding method were from Bio-Rad Laboratories (Hercules, CA).…”

Section: Experimental Section Materialsmentioning

confidence: 99%

Section: Mgl Assaymentioning

confidence: 99%

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Full Mass Spectrometric Characterization of Human Monoacylglycerol Lipase Generated by Large-Scale Expression and Single-Step Purification

et al. 2008

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“…In the first, hydrolysis of 2-AG to arachidonic acid (AA) was quantified by HPLC. 23 Briefly, 2-AG at varying concentrations from 13 to 400 μM and either sol-hMGL (18 ng) or sol-S-hMGL (3.2 μg) in TME buffer (300 μL, total vol) were incubated at 37°C. Reaction samples (50 μL) were taken immediately at the start of the incubation and after 20 min, diluted 1:4 (v/v) with acetonitrile, and centrifuged at 20 000g for 5 min at room temperature.…”

Section: ■ Methodsmentioning

confidence: 99%

Endocannabinoid Enzyme Engineering: Soluble Human Thio-Monoacylglycerol Lipase (sol-S-hMGL)

Karageorgos

Zvonok

Janero

et al. 2012

ACS Chem. Neurosci.

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In the mammalian central nervous system, monoacylglycerol lipase (MGL) is principally responsible for inactivating the endocannabinoid signaling lipid 2-arachidonoylglycerol (2-AG) and modulates cannabinoid-1 receptor (CB1R) desensitization and signal intensity. MGL is also a drug target for diseases in which CB1R stimulation may be therapeutic. To inform the design of human MGL (hMGL) inhibitors, we have engineered a Leu(Leu 169 ;Leu 176 )-to-Ser(Ser 169 ;Ser 176 ) double hMGL mutant (sol-hMGL) which exhibited enhanced solubility properties, and we further mutated this variant by substituting its catalytic-triad Ser 122 with Cys (sol-S-hMGL). The hMGL variants hydrolyzed both 2-AG and a fluorogenic reporter substrate with comparable affinities. Our results suggest that the hMGL cysteine mutant maintains the same overall architecture as wildtype hMGL. The results also underscore the superior nucleophilic nature of the reactive catalytic Ser 122 residue as compared to that of Cys 122 in the sol-S-hMGL mutant and suggest that the nucleophilic character of the Cys 122 residue is not commensurately enhanced within the three dimensional architecture of hMGL. The interaction of the sol-hMGL variants with the irreversible inhibitors AM6580 and N-arachidonylmaleimide (NAM) and the reversible inhibitor AM10212 was profiled. LC/MS analysis of tryptic digests from sol-S-hMGL directly demonstrate covalent modification of this variant by NAM and AM6580, consistent with enzyme thiol alkylation and carbamoylation, respectively. These data provide insight into hMGL catalysis, the key role of the nucleophilic character of Ser 122 , and the mechanisms underlying hMGL inhibition by different classes of small molecules.

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“…Separation and quantification of arachidonic acid product was achieved by gas chromatography [110] and high-performance liquid chromatography (HPLC) [111,112]. HPLC was also used for the separation and detection of released ethanolamine moiety [113].…”

Section: Distributionmentioning

confidence: 99%

Focus on the Three Key Enzymes Hydrolysing Endocannabinoids as New Drug Targets

Vandevoorde¹,

Lambert

2005

CPD

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The family of endocannabinoids (i.e., the endogenous agonists of cannabinoid receptors) contains several polyunsaturated fatty acid amides such as anandamide (AEA) and oleamide but also esters such as 2-arachidonoylglycerol (2-AG). These compounds are the subject of growing interest in pharmacology for their multiple therapeutic potentials. Unfortunately, they are rapidly inactivated by enzymatic hydrolysis, which prevents their effective medical use. Inhibitors of endocannabinoid degradation seem to be necessary tools for the development of endocannabinoid therapeutics. But hitting this target is inconceivable without good knowledge of the enzymes. Fatty acid amide hydrolase (FAAH) is the oldest and the best characterised enzyme involved in the degradation of endocannabinoids. Cloning, distribution in the body and crystal structure of FAAH have been described. A large number of FAAH inhibitors have also been synthesised and tested. For a long time, FAAH was considered as the only key enzyme hydrolysing endocannabinoids. But recent findings indicate that at least two other enzymes have critical role in the endocannabinoids degradation. Monoglyceride lipase participates in 2-AG degradation and some data indicate that it is the primary mechanism for 2-AG inactivation in intact neurons. N-palmitoylethanolamine-selective acid amidase (NPAA) is a second fatty acid amide hydrolase more active with N-palmitoylethanolamine, an anti-inflammatory substance. The purpose of this review is to collect and compare the catalytic properties of these 3 key enzymes hydrolysing endocannabinoids.

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High-Performance Liquid Chromatographic Determination of Anandamide Amidase Activity in Rat Brain Microsomes

Cited by 40 publications

References 0 publications

Full Mass Spectrometric Characterization of Human Monoacylglycerol Lipase Generated by Large-Scale Expression and Single-Step Purification

Full Mass Spectrometric Characterization of Human Monoacylglycerol Lipase Generated by Large-Scale Expression and Single-Step Purification

Endocannabinoid Enzyme Engineering: Soluble Human Thio-Monoacylglycerol Lipase (sol-S-hMGL)

Focus on the Three Key Enzymes Hydrolysing Endocannabinoids as New Drug Targets

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