Clorgyline

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 K_m values (0.22 and 0.14 mM).

AChE (Type VI-S; from Electrophorus electricus), recombinant human MAO-A, MAO-B, and BChE (from equine serum), acetylthiocholine iodide (ATCI), kynuramine, benzylamine, S-butyrylthiocholine iodide (BTCI), 5,5′-dithiobis (2-nitrobenzoic acid) (DTNB), tacrine, donepezil, toloxatone, and lazabemide were purchased from Sigma-Aldrich (St. Louis, MO, USA). Clorgyline and pargyline (irreversible reference inhibitors of MAO-A and MAO-B, respectively) were from BioAssay Systems (Hayward, CA, USA)^{40 (link)}.

Recombinant human MAO-A and MAO-B, kynuramine, benzylamine, toloxatone, lazabemide, AChE (Type VI-S from Electrophorus electricus), BChE (equine serum), acetylthiocholine iodide (ATCI), butyrylthiocholine iodide (BTCI), 5,5′-dithiobis(2-nitrobenzoic acid) (DTNB), tacrine, donepezil, and the BACE-1 activity detection kit were purchased from Sigma-Aldrich (St. Louis, MO, USA). Clorgyline and pargyline were obtained from BioAssay Systems (Hayward, CA, USA).
Roswell Park Memorial Institute-1640 medium (RPMI-1640), Dulbecco’s Modified Eagle Medium (DMEM), fetal bovine serum (FBS), and penicillin/streptomycin solution were purchased from Hyclone Laboratories (San Ramon, CA, USA). The cell counting kit-8 (CCK-8) and dimethyl sulfoxide (DMSO) were obtained from Dojindo Laboratories (Kumamoto, Japan) and Sigma-Aldrich, respectively.