A new assay system for the enzyme, choline kinase (ATP:choline phosphotransferase, EC 2.7.1.32), has been developed. The procedure employed [r4C]choline as the substrate. The product, [14C]phosphorylcholine, was separated from the substrate by a simple 30-min paper chromatography step. After development, the area of the chromatogram which contained the phosphorylcholine was cut out, placed directly into a scintillation cocktail, and counted. This assay was tested on a preparation of partially purified yeast choline kinase. The optimum concentrations of ATP and Mg*+ and the K, for choline were determined and found to agree with published values obtained by other assay methods. The new assay was also shown to be useful in measuring choline kinase activity in primate lung tissue homogenates.
A rapid, simple, accurate, and reproducible assay for the enzyme choline kinase (ATP:choline phosphotransferase, EC 2.7.1.32), capable of measuring activities in tissue homogenates, has not been available. Several dependable methods are presently employed for measuring this enzyme, but all are either time-consuming or subject to interfering reactions.
In 195 1, Kornberg and Pricer (7) developed an elegant assay for kinase activity in which the ADP generated is utilized in two additional enzymatic reactions. The result of these reactions is the disappearance of NADH which can be monitored spectrophotometrically.
This assay, although yielding immediate results, is subject to interfering reactions. Another method (1) involves precipitating the choline remaining after the reaction as the periodide salt and measuring this complex spectrophotometrically. Phosphorylcholine is not precipitated by this method, and, presumably, the amount of choline disappearing in the reaction is proportional to the amount of phosphorylcholine produced. Again, this method could be susceptible to interfering reactions in crude or partially purified enzyme preparations. One isotopic method previously utilized in this laboratory (8) involves the selective precipitation of unreacted choline by ammonium reineckate, leaving the phosphorylcholine in solution to be measured by liquid scintillation counting. This method has proven to be time-consuming and is potentially dangerous since ammonium reineckate can generate hydrogen cyanide. Finally, there are several methods employing ionexchange resins or liquid ion-exchange procedures to separate choline 526