Enzymes (recombinant human MAO-A and MAO-B, AChE from Electrophorus electricus, and BChE from equine serum), substrates (kynuramine and benzylamine, acetylthiocholine iodide (ATCI), S-butyrylthiocholine iodide (BTCI)), inhibitors (toloxatone, lazabemide, and tacrine), and other chemicals including 5,5′-dithiobis(2-nitrobenzoic acid) (DTNB) were purchased from Sigma-Aldrich (St. Louis, MO, USA) [49 (link),58 (link)]. The irreversible inhibitors (clorgyline and pargyline) were obtained from BioAssay Systems (Hayward, CA, USA) [59 (link)].
MAO-A and MAO-B activities were measured continuously at 316 nm for 20 min, and at 250 nm for 30 min, respectively, as described previously [60 (link),61 (link)]. The concentrations used were; kynuramine (0.06 mM) for MAO-A and benzylamine (0.3 mM) for MAO-B. AChE activity was assayed continuously for 10 min at 412 nm using 0.2 U/mL of enzyme in the presence of 0.5 mM DTNB and 0.5 mM ATCI in 0.5 mL of reaction mixture, as previously described [49 (link),58 (link)], based on the method developed by Ellman et al. [62 (link)]. BChE activity was assayed using the same method as AChE, except using BTCI [49 (link)]. Substrate concentrations of BTCI for BChE and benzylamine for MAO-B were 2.3- and 2.1-fold of the respective Km values (0.22 and 0.14 mM).
MAO-A and MAO-B activities were measured continuously at 316 nm for 20 min, and at 250 nm for 30 min, respectively, as described previously [60 (link),61 (link)]. The concentrations used were; kynuramine (0.06 mM) for MAO-A and benzylamine (0.3 mM) for MAO-B. AChE activity was assayed continuously for 10 min at 412 nm using 0.2 U/mL of enzyme in the presence of 0.5 mM DTNB and 0.5 mM ATCI in 0.5 mL of reaction mixture, as previously described [49 (link),58 (link)], based on the method developed by Ellman et al. [62 (link)]. BChE activity was assayed using the same method as AChE, except using BTCI [49 (link)]. Substrate concentrations of BTCI for BChE and benzylamine for MAO-B were 2.3- and 2.1-fold of the respective Km values (0.22 and 0.14 mM).