Partial purification and properties of choline kinase (EC 2. 7. 1. 32) from rabbit brain: measurement of acetylcholine

Choline kinase (EC 2.7.1.32; ATP: choline phosphotransferase) was purified 200-fold from an extract of acetone powder of rabbit brain by a combination of acid precipitation, ammonium sulphate precipitation, DEAE cellulose chromatography, and ultrafiltration. Maximal activity of 243 nmol of phosphorylcholine synthesized. min−1 mg−l of protein occurred at pH 9.5–10.0 in the presence of 10 mm MgS04, 10 mm choline and 0.005% (w/v) bovine serum albumin. 2-Aminoethanol, 2-methylaminoethanol, and 2-dimethylaminoethanol were also phosphorlyated by the enzyme preparation. The enzyme quantitatively converted low concentrations of choline (2.5–50 μm) to phosphorylcholine [32P] in the presence of ATP [y32P], and may, therefore, be used to measure small amounts of choline acetylcholine. There were two Km values for choline at pH 9.5; 32 μm and 0.31 mm. At pH 7.4, the higher Km was not observed and enzyme activity was maximal with 0.1 mm choline. The Km for ATP was 1.1 mm. Enzyme activity was inhibited by ATP (20 mm), AMP, ADP, cytidine diphosphocholine (1 or 10 mm), and activated by choline esters (1.0 mm), NaCl or KCl(200 mm).