Purification of cytidine-triphosphate synthetase from rat liver, and demonstration of monomer, dimer and tetramer

Abstract

Cytidine-triphosphate synthetase (UTP: ammonia ligase (ADP-forming), EC 6.3.4.2.) has been purified over 31 000-fold to homogeneity with 17% recovery from rat liver cytosol, using high-performance liquid chromatography (HPLC) techniques. The presence of CTP synthetase monomer, dimer and tetramer has been demonstrated in the ammonium sulfate fraction of rat liver cytosol. By gel-permeation HPLC, the molecular weights of the three molecular forms of the enzyme have been estimated as 240 000 (tetramer), 120 000 (dimer) and 60 000 (monomer). By gel-permeation chromatography on Bio-Gel A-1.5m column, the molecular weights of dimer and monomer were estimated as 100 000 and 50 000, respectively. The molecular weight of the monomeric subunit is determined to be 66 000 by SDS-polyacrylamide gel electrophoresis. Monomers isolated fresh from 0–30 (NH 4) 2SO 4 fraction of rat liver cytosol are enzymatically active. Purified rat liver CTP synthetase exhibited sigmoidal kinetic plots as a function of the substrate UTP in the presence of the end-product, CTP. Partially purified CTP synthetase usually forms an inactive coagulum on freezing and subsequent thawing. Incubation of CTP synthetase dimer at 25°C for 1 h in the presence of UTP, ATP and Mg 2+ resulted in optimum conversion to tetramer with least inactivation. The purified tetramer dissociates to dimers when UTP, ATP and Mg 2+ are removed by dialysis.