Measurement of choline acetyltransferase with [14C]acetate by a cycling procedure.
A multiple enzyme and multisubstrate cycling system is described for the radiometric determination of cholineacetyltransferase (ChAT) activity in crude tissue homogenates. The methods employs [14C]acetate coupled with the enzymes acetate kinase (AK) and phosphotransacetylase (PTA) for the generation of [14C]acetyl CoA. By recycling it was possible to avoid product inhibition of ChAT by CoA, ATP was maintained constant by rephosphorylation of ADP. Kinetics of the individual enzyme reactions were studied and the parameters obtained were used to select appropriate conditions to maintain linearity of varying amounts ChAT activity over a sixty minute time course. The sensitivity of the method is limited only by the specific activity of commercially available isotope labeled acetate.
D000084:Acetate Kinase; D000085:Acetates; D019342:Acetic Acid; D000105:Acetyl Coenzyme A / Q000378:metabolism; D000818:Animals; D001921:Brain / Q000201:enzymology*; D002250:Carbon Radioisotopes; D002795:Choline O-Acetyltransferase / Q000032:analysis*; D007202:Indicators and Reagents; D007700:Kinetics; D010709:Phosphate Acetyltransferase; D011770:Pyruvate Kinase; D051381:Rats
D000085:Acetates; D002250:Carbon Radioisotopes; D007202:Indicators and Reagents; D000105:Acetyl Coenzyme A; D002795:Choline O-Acetyltransferase; D010709:Phosphate Acetyltransferase; D011770:Pyruvate Kinase; D000084:Acetate Kinase; D019342:Acetic Acid