A single-vial biphasic liquid extraction assay for choline acetyltransferase using [3H]choline
Rand, J.B.; Johnson, C.D.
Analytical Biochemistry 116(2): 361-371
1981
ISSN/ISBN: 0003-2697 PMID: 7316168 Document Number: 174049
A single-vial liquid extraction assay for Caenorhabditis elegans choline acetyltransferase that uses 3H choline as the labeled substrate was devised. 3H choline is incubated with an excess of acetyl-CoA in a small reaction vial which also serves as a scintillation vial. After a suitable reaction period, unreacted 3H choline is quickly and quantitatively converted to phosphoryl- 3H choline by the addition of an excess of brewers yeast choline kinase EC 2.7.1.32 . This treatment is followed by the addition of scintillation fluid containing sodium tetraphenylboron after which the vial is capped, shaken and counted. A 2-phase system is produced in which product 3H acetylcholine is selectively extracted into the scintillation fluid, where it is counted. Phosphoryl- 3H choline remains in the aqueous phase and is not counted. This assay is rapid, simple and quite sensitive. In comparison to assays using acetyl-CoA as the labeled substrate, it is less sensitive to interference by other enzymes and thus more suitable for measuring choline acetyltransferase in crude extracts and in the initial stages of purification. Similar single-vial radiometric assays are described for choline kinase and acetyl-CoA hydrolases.