Elsevier

Life Sciences

Volume 56, Issues 23–24, 5 May 1995, Pages 1999-2005
Life Sciences

Cannabinod receptor and their ligand
Arachidonoyl ethanolamide-[1,2-14C] as a substrate for anandamide amidase

https://doi.org/10.1016/0024-3205(95)00181-5Get rights and content

Abstract

Arachidonoyl ethanolamide-[1,2-14C] was prepared and evaluated as a substrate for anandamide amidase in a radioenzymatic assay that does not require a thin layer chromatography separation step. Using this substrate the release of ethanolamine-[1,2-MC] is linear for approximately thirty minutes. Anandamide amidase exhibits maximal activity between pH 8 and pH 9 with a steep decline in activity at pH values below 6 and above 10. Arachidonoyl ethanolamide-[1,2-14C] was used for the assay of anandamide amidase from 10 μg to 100 μg protein, from cow brain homogenate, in a 0.2 ml incubation mixture. When plotted as a rectangular hyperbola of the steady-state Michaelis-Menten equation, an approximate Km of 30 ± 7 μM and a Vmax of 198 ± 13 nmoles ethanolamine formed per hour per mg protein homogenate was obtained.

References (16)

  • D.G. Deutsch et al.

    Biochem. Pharm.

    (1993)
  • B. Koutek et al.

    J. Biol. Chem.

    (1994)
  • S.R. Childers et al.

    Biochem. Pharm.

    (1994)
  • N.R. Bachur et al.

    J. Biol Chem

    (1966)
  • P.C. Schmid et al.

    J. Biol Chem

    (1985)
  • A.C. Howlett et al.

    Trends Neurosci.

    (1990)
  • L.A. Matsuda et al.

    Nature

    (1990)
  • W.A. Devane et al.

    Science

    (1992)
There are more references available in the full text version of this article.

Cited by (91)

  • Chronic, noninvasive glucocorticoid administration suppresses limbic endocannabinoid signaling in mice

    2012, Neuroscience
    Citation Excerpt :

    values were determined by nonlinear curve fitting of specific binding data to the single site binding equation using GraphPad Prism (San Diego, CA, USA). FAAH activity was measured as the conversion of AEA labeled with [3H] in the ethanolamine portion of the molecule ([3H]AEA; Omeir et al., 1995) to [3H]ethanolamine as reported previously (Hillard et al., 1995). Membranes were incubated in a final volume of 0.5 ml of TME buffer (50 mM Tris–HCl, 3.0 mM MgCl2, and 1.0 mM EDTA, pH 7.4) containing 1.0 mg/ml fatty acid-free bovine serum albumin and 0.2 nM [3H]AEA.

  • Lipid droplets are novel sites of N-acylethanolamine inactivation by fatty acid amide hydrolase-2

    2010, Journal of Biological Chemistry
    Citation Excerpt :

    This suggests that in vitro activities, employing high substrate concentrations, do not reveal the true catabolic capacities of NAE-inactivating enzymes in cells. The elevated activity of FAAH-2 in intact cells is supported by its low Km values for AEA and PEA, which are severalfold lower than those reported for FAAH (25, 26). This suggests higher enzymatic activity at physiological substrate concentrations (i.e. ≤1 μm), such as those used in our intact cell system.

  • Microglia produce and hydrolyze palmitoylethanolamide

    2008, Neuropharmacology
    Citation Excerpt :

    When focusing on PEA hydrolysis by BV-2 cell homogenates, three sets of evidence suggest the involvement of FAAH. First, the pH profile of [3H]-PEA hydrolysis parallels the pH profile described for [3H]-AEA hydrolysis by FAAH, both of which reach optimal activity at pH 8–9 (Omeir et al., 1995; Ueda et al., 1995). Second, MAFP and URB597 inhibit [3H]-PEA hydrolysis with IC50 values corresponding to those reported for recombinant FAAH (Lichtman et al., 2004).

  • Novel inhibitors of fatty acid amide hydrolase

    2007, Bioorganic and Medicinal Chemistry Letters
View all citing articles on Scopus
View full text