Improved radioisotopic assay for cytidine 5'-triphosphate synthetase (EC 6.3.4.2)

An improved radioassay for cytidine 5-triphosphate synthetase is reported which employs thin-layer chromatographic methods and provides a number of advantages over previously available techniques. (i) The method resolves the nucleotides and the degradation products generated during the time course of the enzymatic reaction by ascending chromatography employing polyethyleneimine cellulose plastic-backed sheets. (ii) Determinations of CTP formed and all nucleotide pairs generated during kinetic analysis of CTP synthetase are greatly simplified, further facilitating the detection of extraneous enzymatic activities. (iii) The sensitivity of the assay is enhanced and as little as 50 pmol of product formed was readily detected in supernatant fluids. This was made possible, in part, by the addition of NaF and phosphoenolpyruvate which together maintain the nucleotide triphosphates in the reaction mixture. (iv) A large number of samples can be handled at one time with highly reproducible results. The synthesis of CTP from UTP by enzyme preparations from rat liver, hepatomas, and Salmonella typhimurium LT2 was quantitated with this method.