Purification of cytidine-triphosphate synthetase from rat liver, and demonstration of monomer, dimer and tetramer.
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 (NH4)2SO4 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 degrees C for 1 h in the presence of UTP, ATP and Mg2+ resulted in optimum conversion to tetramer with least inactivation. The purified tetramer dissociates to dimers when UTP, ATP and Mg2+ are removed by dialysis.
Thomas
P E
PE
Department of Human Genetics, University of Michigan Medical School, Ann Arbor 48109-0618.
Lamb
B J
BJ
Chu
E H
EH
D000255:Adenosine Triphosphate / Q000494:pharmacology; D000818:Animals; D019731:Carbon-Nitrogen Ligases; D002850:Chromatography, Gel; D002851:Chromatography, High Pressure Liquid; D003600:Cytosol / Q000201:enzymology; D004591:Electrophoresis, Polyacrylamide Gel; D005260:Female; D005590:Fractional Precipitation; D007700:Kinetics; D008025:Ligases / Q000302:isolation & purification*; D008099:Liver / Q000201:enzymology*; D046911:Macromolecular Substances; D008274:Magnesium / Q000494:pharmacology; D008970:Molecular Weight; D051381:Rats; D011919:Rats, Inbred Strains; D014544:Uridine Triphosphate / Q000494:pharmacology
D046911:Macromolecular Substances; D000255:Adenosine Triphosphate; D008025:Ligases; D019731:Carbon-Nitrogen Ligases; C019951:CTP synthetase; D008274:Magnesium; D014544:Uridine Triphosphate