A Procedure for the Microdetermination of Choline Acetyltransferase
Technical rept. Mar-Sep 1971
EDGEWOOD ARSENAL ABERDEEN PROVING GROUND MD
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A micromethod for the determination of choline acetyltransferase was developed using 300 micro M acetyl Co A and 5 mM choline as substrates. The reaction yielded 14C acetylcholine, which was extracted with sodium tetraphenylboron in 3-heptanone and an aliquot was counted utilizing a Tricarb scintillation spectrometer. The sensitivity of the method was approximately 10 to the -12th power moles of 14C acetylcholine synthesizedhour. Using whole rat brain homogenates, the specific activity was determined after the addition of Triton X-100 0.5 to the homogenate in 0.32 M sucrose. Soman 0.1 mM did not inhibit choline acetyltransferase activity however, a specific tertiary inhibitor of choline acetyltransferase 4-1-naphthylvinyl-pyridine 0.1 mM, was effective in producing 100 inhibition. A brief study was conducted using various inhibitors of choline acetyltransferase in order to determine the possibility of regulating acetylcholine synthesis and also to determine the possibility of purifying the enzyme utilizing affinity chromatography.