Phenyldichlorophosphate as an aid in studies of decarbamylation of carbamylated acetylcholinesterase.
An improved method for assaying carbamylated acetylcholinesterase is described which has substantial benefits over current methods. Acetylcholinesterase was carbamylated with neostigmine and diluted extensively into buffer to allow decarbamylation to occur. At various times, phenyldichlorophosphate was added to the mixture of free and carbamylated enzyme, whereupon two very rapid, simultaneous reactions occurred: near total, and permanent, inactivation of free acetylcholinesterase by the organophosphate, and inactivation of phenyldichlorophosphate by hydrolysis. The carbamylated acetylcholinesterase was allowed to reactivate fully and then assayed for enzyme activity. The assay provided a measure of the amount of carbamylated enzyme present at the time of addition of phenyldichlorophosphate, thereby enabling the first-order rate constant for decarbamylation to be calculated. This new method of studying decarbamylation was applied to two systems of soluble acetylcholinesterase, where the half-life for decarbamylation was approximately 1/2 h or 4 min, respectively, and to membrane-bound acetylcholinesterase. The results agreed well with those determined by a conventional method; moreover, the standard error of the mean was lower for the new method. The advantages of the method using phenyldichlorophosphate over conventional methods are particularly evident when decarbamylation is rapid or when in vivo studies are being performed and it is not practical or desirable to run assays immediately on isolation of the tissue. The new method also has advantages over a published related technique using the organophosphate anticholinesterase soman.
Dawson
R M
RM
Materials Research Laboratories, Defence Science and Technology Organisation, Victoria, Australia.
Poretski
M
M
D000110:Acetylcholinesterase / Q000378:metabolism*; D000818:Animals; D002219:Carbamates / Q000378:metabolism*; D002800:Cholinesterase Inhibitors / Q000378:metabolism*; D004593:Electrophorus; D004910:Erythrocyte Membrane / Q000201:enzymology; D006168:Guinea Pigs; D009943:Organophosphorus Compounds / Q000494:pharmacology*; D012995:Solubility
D002219:Carbamates; D002800:Cholinesterase Inhibitors; D009943:Organophosphorus Compounds; C001036:dimethylcarbamyl chloride; C056491:phenyl dichlorophosphate; D000110:Acetylcholinesterase