Highly sensitive assay for choline acetyltransferase activity by high-performance liquid chromatography with electrochemical detection.
A highly sensitive assay for choline acetyltransferase activity by high-performance liquid chromatography with electrochemical detection was devised. This assay method is based on the separation of acetylcholine and choline on a Develosil Ph-5 reversed-phase column (a phenyl column), followed by their enzymatic conversion to hydrogen peroxide through post-column reaction with acetylcholinesterase and choline oxidase. The sensitivity of the system is excellent and 5 pmol of acetylcholine enzymatically formed could be detected. The linearity between the peak height and the amount of acetylcholine was observed over the range of 5 pmol to 5 nmol. Some enzymatic properties were investigated by using a soluble fraction of bovine caudate nucleus as enzyme. The Michaelis constants of the enzyme for choline and acetyl coenzyme A were 0.3 mM and 0.03 mM, respectively. The enzyme exhibited the maximum activity over the pH range 7.4-9.5. The regional distribution of choline acetyltransferase activity in rat brain was examined. The order of the activity from the highest to the lowest agreed with the reported brain distribution of the enzyme: striatum, pons plus medulla oblongata, cerebral cortex, thalamus plus hypothalamus, olfactory bulb and cerebellum.
D000109:Acetylcholine / Q000032:analysis; D000110:Acetylcholinesterase / Q000032:analysis; D000818:Animals; D001921:Brain / Q000201:enzymology*; D002417:Cattle; D002421:Caudate Nucleus / Q000201:enzymology; D002794:Choline / Q000032:analysis; D002795:Choline O-Acetyltransferase / Q000032:analysis*; D002851:Chromatography, High Pressure Liquid; D004563:Electrochemistry; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D010830:Physostigmine / Q000032:analysis; D051381:Rats
D010830:Physostigmine; D002795:Choline O-Acetyltransferase; D000110:Acetylcholinesterase; D002794:Choline; D000109:Acetylcholine