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

Thomas, Peedikayil E.; Lamb, Barbara; Chu, Ernest H. Y.

doi:10.1016/0167-4838(88)90042-8

Cited by 27 publications

(31 citation statements)

References 30 publications

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“…CTP inhibits the CTP synthetase activity by increasing the positive cooperativity of the enzyme for UTP, and at the same time, decreasing the affinity (as reflected in an increase in K m ) for UTP [21,22]. This inhibition mechanism is also true for the CTP synthetases from E. coli [2] and rat liver [26]. Because CTP and UTP have distinct binding sites on CTP synthetase [32,40], it is likely that CTP does not compete with UTP for the active site.…”

Section: Ctp Regulates Ctp Synthetase Activity and The Synthesis Of Mmentioning

confidence: 92%

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CTP synthetase and its role in phospholipid synthesis in the yeast Saccharomyces cerevisiae

Chang

Carman

2008

Progress in Lipid Research

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CTP synthetase is a cytosolic-associated glutamine amidotransferase enzyme that catalyzes the ATPdependent transfer of the amide nitrogen from glutamine to the C-4 position of UTP to form CTP. In the yeast Saccharomyces cerevisiae, the reaction product CTP is an essential precursor of all membrane phospholipids that are synthesized via the Kennedy (CDP-choline and CDP-ethanolamine branches) and CDP-diacylglycerol pathways. The URA7 and URA8 genes encode CTP synthetase in S. cerevisiae, and the URA7 gene is responsible for the majority of CTP synthesized in vivo. The CTP synthetase enzymes are allosterically regulated by CTP product inhibition. Mutations that alleviate this regulation result in an elevated cellular level of CTP and an increase in phospholipid synthesis via the Kennedy pathway. The URA7-encoded enzyme is phosphorylated by protein kinases A and C, and these phosphorylations stimulate CTP synthetase activity and increase cellular CTP levels and the utilization of the Kennedy pathway. The CTPS1 and CTPS2 genes that encode human CTP synthetase enzymes are functionally expressed in S. cerevisiae, and rescue the lethal phenotype of the ura7Δ ura8Δ double mutant that lacks CTP synthetase activity. The expression in yeast has revealed that the human CTPS1-encoded enzyme is also phosphorylated and regulated by protein kinases A and C.

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Section: Ctp Regulates Ctp Synthetase Activity and The Synthesis Of Mmentioning

confidence: 92%

“…CTP synthetase enzymes have been isolated from both prokaryotic and eukaryotic organisms [2,21,22,[24][25][26][27]. Moreover, structures for the bacterial [28,29] and human [30] enzymes have been solved.…”

Section: Ctp Synthetases Of S Cerevisiaementioning

confidence: 99%

CTP synthetase and its role in phospholipid synthesis in the yeast Saccharomyces cerevisiae

Chang

Carman

2008

Progress in Lipid Research

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“…CTP synthetase has been purified and characterized from bacteria (21)(22)(23), Saccharomyces cerevisiae (8,24), and rat liver (25). In addition, crystal structures for the Escherichia coli (26) and Thermus thermophilus (27) enzymes have been solved.…”

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confidence: 99%

“…2 An important mode of CTP synthetase regulation is feedback inhibition by CTP (8,21,(23)(24)(25). CTP inhibits CTP synthetase activity by increasing the positive cooperativity of the enzyme for UTP (8,21,24,25). dCTP does not substitute for CTP as a feedback inhibitor of CTP synthetase activity using dUTP or UTP as a substrate (23,36).…”

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Expression of Human CTP Synthetase in Saccharomyces cerevisiae Reveals Phosphorylation by Protein Kinase A

Han

Sreenivas

Choi

et al. 2005

Journal of Biological Chemistry

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CTP synthetase (EC 6.3.4.2, UTP:ammonia ligase (ADP-forming)) is an essential enzyme in all organisms; it generates the CTP required for the synthesis of nucleic acids and membrane phospholipids. In this work we showed that the human CTP synthetase genes, CTPS1 and CTPS2, were functional in Saccharomyces cerevisiae and complemented the lethal phenotype of the ura7⌬ ura8⌬ mutant lacking CTP synthetase activity. The expression of the CTPS1-and CTPS2-encoded human CTP synthetase enzymes in the ura7⌬ ura8⌬ mutant was shown by immunoblot analysis of CTP synthetase proteins, the measurement of CTP synthetase activity, and the synthesis of CTP in vivo. Phosphoamino acid and phosphopeptide mapping analyses of human CTP synthetase 1 isolated from 32 P i -labeled cells revealed that the enzyme was phosphorylated on multiple serine residues in vivo. Activation of protein kinase A activity in yeast resulted in transient increases (2-fold) in the phosphorylation of human CTP synthetase 1 and the cellular level of CTP. Human CTP synthetase 1 was also phosphorylated by mammalian protein kinase A in vitro. Using human CTP synthetase 1 purified from Escherichia coli as a substrate, protein kinase A activity was dose-and time-dependent, and dependent on the concentrations of CTP synthetase 1 and ATP. These studies showed that S. cerevisiae was useful for the analysis of human CTP synthetase phosphorylation.CTP synthetase (EC 6.3.4.2, UTP:ammonia ligase (ADP-forming)) catalyzes the final step in the pyrimidine biosynthetic pathway (1). The end product CTP is required for the synthesis of nucleic acids and membrane phospholipids (2). Thus, CTP synthetase is an essential enzyme for the growth and metabolism of all organisms (2). In eukaryotes, CTP synthetase activity regulates the balance of nucleotide pools (3-9) and influences the pathways by which membrane phospholipids are synthesized (9 -11). The importance of understanding the mode of action and regulation of CTP synthetase is further highlighted by the fact that elevated CTP synthetase activity is a common property of several cancers in humans (12)(13)(14)(15)(16)(17)(18)(19)(20).CTP synthetase has been purified and characterized from bacteria (21-23), Saccharomyces cerevisiae (8, 24), and rat liver (25). In addition, crystal structures for the Escherichia coli (26) and Thermus thermophilus (27) enzymes have been solved. The enzymological properties of CTP synthetase enzymes from various sources are similar, although some differences have been identified (23). The enzyme catalyzes a complex set of reactions involving the ATP-dependent transfer of the amide nitrogen from glutamine (i.e. glutaminase reaction) to the C-4 position of UTP to generate CTP (Fig. 1) (21, 28). GTP activates the glutaminase reaction by accelerating the formation of a covalent glutaminyl enzyme intermediate (21,29). CTP synthetase exhibits positive cooperative kinetics with respect to UTP and ATP and negative cooperative kinetics with respect to glutamine and GTP (8, 21, 23, 24, 29 -33). The positive c...

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Molecular cloning of the human CTP synthetase gene by functional complementation with purified human metaphase chromosomes.

Yamauchi¹,

Yamauchi²,

Meuth³

1990

The EMBO Journal

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Successive rounds of chromosome‐mediated gene transfer were used to complement a hamster cytidine auxotroph deficient in CTP synthetase activity and eventually to clone human genomic and cDNA fragments coding for the structural gene. Our approach was to isolate human Alu+ fragments from a tertiary transfectant and to utilize these fragments to screen a panel of primary transfectants. In this manner two DNA fragments, both mapping within the structural gene, were identified and used to clone a partial length cDNA. The remaining portion of the open reading frame was obtained through the RACE polymerase chain reaction technique. The open reading frame encodes 591 amino acids having a striking degree of similarity to the Escherichia coli structural gene (48% identical amino acids with 76% overall similarity including conservative substitutions) with the glutamine amide transfer domain being particularly conserved. As regulatory mutations of CTP synthetase confer both multi‐drug resistance to agents widely used in cancer chemotherapy and a mutator phenotype, the cloning of the structural gene will be important in assessing the relevance of such phenotypes to the development of cellular drug resistance.

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

Cited by 27 publications

References 30 publications

CTP synthetase and its role in phospholipid synthesis in the yeast Saccharomyces cerevisiae

CTP synthetase and its role in phospholipid synthesis in the yeast Saccharomyces cerevisiae

Expression of Human CTP Synthetase in Saccharomyces cerevisiae Reveals Phosphorylation by Protein Kinase A

Molecular cloning of the human CTP synthetase gene by functional complementation with purified human metaphase chromosomes.

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