Acetylthiocholine

For the in vitro studies, tetrameric CHO-derived rMaBChE and rHuBChE were produced and purified using procainamide Sepharose chromatography as previously described [18,29]. RBC Ma-AChE was prepared from 7.5 ml of rhesus macaque blood, which was centrifuged at 1,000 RPM for 15 min. Collected RBCs were washed three times with PBS before being lysed in water to a final volume of 4 ml. Recombinant HuAChE was produced in HEK 293 cells (27 (link), 28 (link)).
BChE and AChE activity was assayed using 1 mM butyrylthiocholine or acetylthiocholine (Sigma-Aldrich) and 0.5 mM 5,5-dithiobis 2-nitrobenzoic acid (DTNB) in 50 mM sodium phosphate buffer, pH 8.0, at 22°C. The formation of product was followed by monitoring the increase in absorbance of 5-thio-2- nitrobenzoic acid at 412 nm using a molar extinction coefficient of 13,600 M⁻¹. Activity was reported as U/ml where 1 U represents 1 µmole of acetyl- or butyrylthiocholine per min. In the AChE activity assay, 20 µM ethopropazine was used as a BChE-specific inhibitor. The specific activity of MaBChE (900 U/mg) is slightly higher than HuBChE (700 U/mg). Background levels in macaque blood generally range from 2.5 to 6.5 U/ml AChE and 3.5 to 8.5 U/ml BChE.

Plasma ChE as well as RBC and CNS AChE activities were determined via a modification of the Ellman method as previously described (Chambers and Chambers, 1989 (link), 1990 (link); Ellman et al., 1961 (link); Kondakala et al., 2017 (link)). Briefly, RBCs were diluted to a final concentration of 0.0005 μl RBC/ml in sodium phosphate buffer (0.1 M, pH 7.4) and absorbance monitored continuously after addition of substrates for 60 min at 412 nm. Brain tissue was homogenized in 0.05 M Tris-HCl (pH 7.4) and homogenates were then pre-incubated at 37 °C for 15 min. Acetylthiocholine (1 mM final concentration; Sigma Aldrich) was then added as a substrate and the reaction continued for an additional 15 min. At the end of 15 min, the reaction was terminated by addition of 250 μl of a 4:1 (v:v) mixture of 5% SDS:5,5′-dithio-bis-(2-nitrobenzoic acid) (DTNB) then absorbance measured at 412 nm. Protein concentrations were measured by Bradford assay and data expressed as nanomoles (nmoles) per minute per mg protein.