EVALUATION OF THE NATURE OF RAT RETINAL ACETYLCHOLINESTERASE USING A SPECIFIC SUBSTRATE AND A SPECIFIC INHIBITOR

The occurrence of cholinesterases (ChE) has been demonstrated in retin as of several mammalian species. Using BW284C51 and iso-OMPA as select ive inhibitors of acetylcholinesterase (AChE) and butyrylcholinesteras e (BChE), respectively, it has been demonstrated that the rat retinal ChE is predominantly AChE. Therefore the kinetic nature of inhibition of the rat retinal AChE by BW284C51 was studied using acetyl-beta-meth ylthiocholine (AMTCh) as a selective substrate of AChE. AChE activity of the rat retinal sonicates was assayed using AMTCh as the substrate in the presence of 5,5-dithiobis-2-nitrobenzoate and yellow 5-thio-2-n itrobenzoic anion was measured by the absorption at 412 m mu using a s pectrophotometer. The substrate (AMTCh) was varied between 0.1 and 0.5 mM. The inhibitor concentrations used were 2.1 and 4.2 nM. Double-rec iprocal plots between substrate concentrations and the velocities for the enzymatic hydrolysis of AMTCh in the presence and absence of inhib itor were constructed. This study gave the following results: BW284C51 was a potent inhibitor of the hydrolysis of AMTCh by rat retinal AChE (IC50, 5.2 nM). The nature of the inhibition was found to be competit ive as the double reciprocal plots with and without the inhibitor cros sed on the ordinate.