Table 1

Effect of DAK on kinetics of AEA amidohydrolase with [3H]AEA as a substrate

TreatmentIntact Membrane1-aDetergent-Solubilized1-b
Km1-cVmax1-cKm1-cVmax1-c
μM μM
DMSO2.0  ± 0.31.2  ± 0.17.5  ± 2.57.5  ± 0.8
1 nM DAK1.9  ± 0.61.3  ± 0.14.5  ± 1.26.3  ± 0.6
10 nM DAK2.5  ± 0.50.9  ± 0.16.0  ± 2.27.7  ± 1.0
100 nM DAK12.2  ± 4.40.4  ± 0.136  ± 106.1  ± 0.5
1 μM DAKn.d.n.d.318  ± 141-d 7.01-d

  • ↵1-a Forebrain membranes were preincubated with DAK or DMSO for 60 min before the addition of AEA. AEA amidohydrolase activity was determined at eight concentrations of AEA between 0.01 nM and 30 μM; the concentration of [3H]AEA was kept constant at 0.01 nM, and the total concentration of AEA was adjusted with the addition of unlabeled AEA.

  • ↵1-b Forebrain membranes were solubilized with Triton X-100 and then incubated with DAK for 60 min before the addition of AEA. AEA amidohydrolase activity was determined at eight concentrations of AEA between 0.01 nM and 300 μM; the concentration of [3H]AEA was kept constant at 0.01 nM and the total concentration of AEA was adjusted with the addition of unlabeled AEA.

  • ↵1-c Km and Vmax were determined from saturation isotherms using nonlinear curve fitting. Units for Vmax are nmol/min/mg protein. Parameters are shown ± S.E.M.

  • ↵1-d Vmax was fixed at 7.0 nmol/min/mg protein.