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Purine Biosynthesis (purine + biosynthesis)

Distribution by Scientific Domains
Life Sciences66%
Chemistry33%

Selected Abstracts

Methotrexate induced differentiation in colon cancer cells is primarily due to purine deprivation

JOURNAL OF CELLULAR BIOCHEMISTRY, Issue 1 2006
R. Singh
Abstract The folate antagonist methotrexate (MTX) inhibits synthesis of tetrahydrofolate (THF), pyrimidines and purines, and induces differentiation in several cell types. At 1 µM, MTX reduced proliferation and induced differentiation in HT29 colon cancer cells; the latter effect was augmented (P,<,0.001) by thymidine (100 µM) but was reversed (P,<,0.001) by the purines, hypoxanthine (Hx; 100 µM) and adenosine (100 µM). In contrast 5-fluoro-uracil (5-FU), a specific thymidylate synthase (TS) inhibitor, had no effect on differentiation, suggesting that MTX-induced differentiation is not due to a reduction in thymidine but to the inhibition of purine biosynthesis. Inhibition of cyclic AMP (cAMP) by RpcAMP (25 µM) further enhanced (P,<,0.001) MTX induced differentiation, whereas the cAMP activator forskolin (10 µM) reversed (P,<,0.001) MTX induced differentiation. These observations implicate a central role of adenosine and cAMP in MTX induced differentiation. By combining Western blot analysis with liquid chromatography-mass spectrometry (LC-MS)and HPLC analyses we also reveal both the expression and activity of key enzymes (i.e. methionine synthase (MS), s-adenosylhomocysteinase, cystathionine ,-synthase and ornithine decarboxylase) regulating methyl cycle, transsulfuration and polyamine pathways in HT29 colon cancer cells. At 1 µM, MTX induced differentiation was associated with a marked reduction in the intracellular concentrations of adenosine and, consequently, S-adenosylmethionine (SAM), S-adenosylhomocysteine, polyamines and glutathione (GSH). Importantly, the marked reduction in methionine that accompanied MS inhibition following MTX treatment was non-limiting with respect to SAM synthesis. Collectively, these findings indicate that the effects of MTX on cellular differentiation and single carbon metabolism are primarily due to the intracellular depletion of purines. J. Cell. Biochem. © 2006 Wiley-Liss, Inc. [source]

Regulation of purine biosynthesis by a eukaryotic-type kinase in Streptococcus agalactiae

MOLECULAR MICROBIOLOGY, Issue 5 2005
Lakshmi Rajagopal
Summary Group B streptococci (GBS) are the principal causal agents of human neonatal pneumonia, sepsis and meningitis. We had previously described the existence of a eukaryotic-type serine/threonine kinase (Stk1) and phosphatase (Stp1) in GBS that regulate growth and virulence of the pathogen. Our previous results also demonstrated that these enzymes reversibly phosphorylated an inorganic pyrophosphatase. To understand the role of these eukaryotic-type enzymes on growth of GBS, we assessed the stk1 -mutants for auxotrophic requirements. In this report, we describe that in the absence of the kinase (Stk1), GBS are attenuated for de novo purine biosynthesis and are consequently growth arrested. During growth in media lacking purines, the intracellular G nucleotide pools (GTP, GDP and GMP) are significantly reduced in the Stk1-deficient strains, while levels of A nucleotides (ATP, ADP and AMP) are marginally increased when compared with the isogenic wild-type strain., We, provide, evidence, that, the, reduced, pools of, G, nucleotides, result, from, altered, activity, of, the IMP utilizing enzymes, adenylosuccinate synthetase (PurA) and IMP dehydrogenase (GuaB) in these strains. We also demonstrate that Stk1 and Stp1 reversibly phosphorylate and consequently regulate PurA activity in GBS. Collectively, these data indicate the novel role of eukaryotic-type kinases in regulation of metabolic processes such as purine biosynthesis. [source]

Crystallization and preliminary X-ray diffraction analysis of recombinant hydrolase domain of 10-­formyltetrahydrofolate dehydrogenase

ACTA CRYSTALLOGRAPHICA SECTION D, Issue 10-2 2002
Alexander A. Chumanevich
10-Formyltetrahydrofolate dehydrogenase (FDH) is an abundant enzyme in liver cytosol. It is important for the regulation of 10-­formyltetrahydrofolate/tetrahydrofolate pools, for de novo purine biosynthesis and for the removal of formate in the form of CO2. The enzyme is a natural fusion of two unrelated genes and consists of two functional catalytic domains. Here, the crystallization of the N-­terminal domain of FDH is reported. This domain binds folate and functions as a 10-formyltetrahydrofolate hydrolase. The crystals grow as either spear-shaped needles or large plates, with the largest crystals reaching dimensions of 1.2 × 0.2 × 0.05,mm. Diffraction analysis revealed the space group to be P21212, with unit-cell parameters a = 100.00, b = 64.63, c = 64.59,Å. Based on the estimated solvent content, there is one 34,kDa molecule in the asymmetric unit. A native data set extending to 2.3,Å resolution has been collected with good merging statistics. [source]

Cloning, expression, purification, crystallization and preliminary X-ray crystallographic study of the putative SAICAR synthetase (PH0239) from Pyrococcus horikoshii OT3

ACTA CRYSTALLOGRAPHICA SECTION F (ELECTRONIC), Issue 2 2010
Kavyashree Manjunath
The study of proteins involved in de novo biosynthesis of purine nucleotides is central in the development of antibiotics and anticancer drugs. In view of this, a protein from the hyperthermophile Pyrococcus horikoshii OT3 was isolated, purified and crystallized using the microbatch method. Its primary structure was found to be similar to that of SAICAR synthetase, which catalyses the seventh step of de novo purine biosynthesis. A diffraction-quality crystal was obtained using Hampton Research Crystal Screen II condition No. 34, consisting of 0.05,M cadmium sulfate hydrate, 0.1,M HEPES buffer pH 7.5 and 1.0,M sodium acetate trihydrate, with 40%(v/v) 1,4-butanediol as an additive. The crystal belonged to space group P31, with unit-cell parameters a = b = 95.62, c = 149.13,Å. Assuming the presence of a hexamer in the asymmetric unit resulted in a Matthews coefficient (VM) of 2.3,Å3,Da,1, corresponding to a solvent content of about 46%. A detailed study of this protein will yield insights into structural stability at high temperatures and should be highly relevant to the development of antibiotics and anticancer drugs targeting the biosynthesis of purine nucleotides. [source]