Abstract
The regulatory role of the allosteric site of CTP synthetase on flux through the enzyme in situ and on pyrimidine nucleotide triphosphate (NTP) pool balance was investigated using a mutant mouse T lymphoblast (S49) cell line which contains a CTP synthetase refractory to complete inhibition by CTP. Measurements of [3H]uridine incorporation into cellular pyrimidine NTP pools as a function of time indicated that CTP synthesis in intact wild type cells was markedly inhibited in a cooperative fashion by small increases in CTP pools, whereas flux across the enzyme in mutant cells was much less affected by changes in CTP levels. The cooperativity of the allosteric inhibition of the enzyme was greater in situ than in vitro. Exogenous manipulation of levels of GTP, an activator of the enzyme, indicated that GTP had a moderate effect on enzyme activity in situ, and changes in pools of ATP, a substrate of the enzyme, had small effects on CTP synthetase activity. The consequences of incubation with actinomycin D, cycloheximide, dibutyryl cyclic AMP, and 6-azauridine on the flux across CTP synthetase and on NTP pools differed considerably between wild type and mutant cells. Under conditions of growth arrest, an intact binding site for CTP on CTP synthetase was required to maintain a balance between the CTP and UTP pools in wild type cells. Moreover, wild type cells failed to incorporate H14CO3- into pyrimidine pools following growth arrest. In contrast, mutant cells incorporated the radiolabel at a high rate indicating loss of a regulatory function. These results indicated that uridine nucleotides are important regulators of pyrimidine nucleotide synthesis in mouse S49 cells, and CTP regulates the balance between UTP and CTP pools.
Highlights
Theregulatoryrole of the allostericsite of CTP quently, the CTP pools are markedly elevated
Mutant cells incorporated the radiolabel at a high rate indicatingloss of a regulatory function.These results indicated that uridine nucleotides are important regulatorsof pyrimidine nucleotide synthesis in mouse S49 cells, and CTP regulates the balance between UTP and CpToPols
Studieson C T P synthetase are of particular interest, since the enzyme has served as a model for allosteric regulation
Summary
From the $Children's Hospital Research Foundation, University of Cincinnati Medical School,Cincinnati, Ohio 45229 and the §Department of Biochemistry, The Oregon Health Sciences University,Portland, Oregon 97201. Lysis in 0.5 N perchloric acid followedby neutralization by the method Effectors of Cell Growth-The growth of exponentially dividing of Khym [11].NTPs were separated by high pressure liquid cells was arrested by three different regimens: a 4-6 h exposure to 40 chromatography (HPLC) with astronganion exchange column pg of cycloheximide/ml [16, 17], and 18-h exposure to 0.5 mM dibu-. 94% of the total perchloric acid-soluble cytidine-containing nucleo- Rates of Nucleotide Biosynthesis-The rates of purine and pyrimitides in wild type and FURT-1A cells, respectively [7]. CTP and dine biosynthesis in wild type and FURT-1A cells were determined dCTP were collected in the same fraction in which they were co- by measuring the rates of H14C03incorporation intoNTPs. 10s eluted, whereas the UTPfraction was collectedseparately.
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