Human cytidine triphosphate synthetase 1 interacting proteins

We investigated the interacting proteins and intracellular localization of CTP synthetase 1 (CTPS1) in mammalian cells. CTPS1 interacted with a GST- peptidyl prolyl isomerase, Pin1 fusion (GST-Pin1) in a Ser 575 (S575) phosphorylation-dependent manner. Immunoprecipitation experiments demonstrated that CTPS1 also bound tubulin, and thirteen additional coimmunoprecipitating proteins were identified by mass spectrometry. Immunolocalization experiments showed that tubulin and CTPS1 colocalized subcellularly. Taxol treatment enhanced this but cotreatment of cells with the CTPS inhibitor, cyclopentenyl cytosine (CPEC), and taxol failed to disrupt the colocalization. Thus, these studies provide novel information on the potential interacting proteins that may regulate CTPS1 function or intracellular localization.     

Leukotriene formation by bovine polymorphonuclear leukocytes

The leukotriene production by bovine polymorphonuclear leukocytes isolated from peripheral blood has been studied. Cells were incubated in the presence of arachidonic acid, glutathione, calcium ionophore A23187 and Ca2+. The leukotrienes then formed are leukotriene C4, leukotriene B4, two all-trans isomers of leukotriene B4 and the double dioxygenation product 12-epi-6-trans-8-cis-leukotriene B4. Leukotriene C4 is formed in such a large quantity by the bovine polymorphonuclear leukocyte that it might constitute an excellent and inexpensive source for the biosynthetic preparation of this spasmogenic leukotriene.     

Aromatase (estrogen synthetase) gene expression in human leukocytes

Extragonadal aromatization plays an important role in many physiological and pathological conditions. It has been found that incubation of the peripheral blood mononuclears in the RPMI-1640 medium with 10% fetal calf serum during 48 hrs is able to induce the aromatase gene expression. The latter did not occur in 11 samples of mononuclears of the intra-tissue leukocyte infiltration which finding needs further investigation. The data obtained suggest that the possibility of the aromatase gene induction in leukocytes is associated with the cellular fraction capable of the substrate adhesion.     

Regulation of human cytidine triphosphate synthetase 1 by glycogen synthase kinase 3

Cytidine triphosphate synthetase (CTPS) catalyzes the rate-limiting step in the de novo synthesis of CTP, and both the yeast and human enzymes have been reported to be regulated by protein kinase A or protein kinase C phosphorylation. Here, we provide evidence that stimulation or inhibition of protein kinase A and protein kinase C does not alter the phosphorylation of endogenous human CTPS1 in human embryonic kidney 293 cells under the conditions tested. Unexpectedly, we found that low serum conditions increased phosphorylation of endogenous CTPS1 and this phosphorylation was inhibited by the glycogen synthase kinase 3 (GSK3) inhibitor indirubin-3'-monoxime and GSK3beta short interfering RNAs, demonstrating the involvement of GSK3 in phosphorylation of endogenous human CTPS1. Separating tryptic peptides from [(32)P]orthophosphate-labeled cells and analyzing the phosphopeptides by mass spectrometry identified Ser-574 and Ser-575 as phosphorylated residues. Mutation of Ser-571 demonstrated that Ser-571 was the major site phosphorylated by GSK3 in intact human embryonic kidney 293 cells by GSK3 in vitro. Furthermore, mutation of Ser-575 prevented the phosphorylation of Ser-571, suggesting that phosphorylation of Ser-575 was necessary for priming the GSK3 phosphorylation of Ser-571. Low serum was found to decrease CTPS1 activity, and incubation with the GSK3 inhibitor indirubin-3'-monoxime protected against this decrease in activity. Incubation with an alkaline phosphatase increased CTPS1 activity in a time-dependent manner, demonstrating that phosphorylation inhibits CTPS1 activity. This is the first study to investigate the phosphorylation and regulation of human CTPS1 in human cells and suggests that GSK3 is a novel regulator of CTPS activity.     

Repression of cytidine triphosphate synthetase in Salmonella typhimurium by pyrimidines during uridine nucleotide depletion

Regulation of the synthesis of cytidine triphosphate (CTP) synthetase (EC 6.3.4.2) was investigated in Salmonella typhimurium. CTP synthetase appeared to be repressed only when intracellular concentrations of uridine nucleotides were significantly lowered. Under such nucleotide pool conditions, a cytidine compound and, to a lesser degree, a thymidine compound appeared as putative repressing metabolites of enzyme synthesis.     

Cyclic cytidine monophosphate stimulates DNA synthesis by bovine mammary tissue in vitro

Mammary gland biopsies were taken from midpregnant heifers (n = 4), cut into pieces .5 mm thick and 3 - 5 mm2 and incubated for 48 hours in Eagle's Minimum Essential Medium containing 0, .1 or 1 micrograms/ml insulin and 0, 10(-8), 10(-7), 10(-6), 10(-5), or 10(-4) M dibutyryl cyclic 3', 5', cytidine monophosphate (dbcCMP). With 0 or .1 microgram/ml insulin, dbcCMP decreased incorporation of tritiated thymidine into DNA. Similar declines in DNA synthesis were observed with sodium butyrate, suggesting that the decline was due to the butyrate rather than to a cyclic CMP-specific effect. With 1 micrograms/ml insulin, dbcCMP increased DNA synthesis. Higher levels of dbcCMP reduced DNA synthesis relative to 10(-6)M dbcCMP, as did sodium butyrate. Thus cCMP is capable of stimulating mammary growth.     

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.     

A high-performance liquid chromatography method for the assay of cytidine monophosphate-sialic acid synthetase

An HPLC method has been developed for the assay of cytidine monophosphate-sialic acid synthetase (EC 2.7.7.43) using ion-pair chromatography and gradient elution. This procedure permits the assay of alternative substrates and inhibitors of the enzyme and is not subject to the limitation of the colorimetric method. The newly synthesized N-acetyl-9-deoxy-9-fluoro-D-neuraminic acid was found to be a good substrate of the enzyme with a Km of 6.35 mM as compared to 1.84 mM for N-acetylneuraminic acid.