High-performance liquid column chromatography of nucleotides, nucleosides and bases

Zakaria, Mona; Brown, Phyllis R.

doi:10.1016/s0378-4347(00)86062-4

Cited by 164 publications

(23 citation statements)

References 52 publications

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“…The HPLC eluate was divided into 1-ml fractions that were individually mixed with scintillant and counted using a PerkinElmer liquid scintillation counter. For assays of adenosine nucleotides, perchloric acid-quenched extracts (Choi et al, 2005) were resolved by HPLC using a 0.46 ϫ 25 cm Vydac 3021C HPLC column (GraceVydac, Hesperia, CA) (Zakaria and Brown, 1981). The [ATP] was directly measured from the absorbance at 260 nm.…”

Section: Measurements Of Cellular Levels Of Inositol Phosphates and Amentioning

confidence: 99%

Cellular Energetic Status Supervises the Synthesis of Bis-Diphosphoinositol Tetrakisphosphate Independently of AMP-Activated Protein Kinase

Choi¹,

Mollapour²,

Choi³

et al. 2008

Mol Pharmacol

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Cells aggressively defend adenosine nucleotide homeostasis; intracellular biosensors detect variations in energetic status and communicate with other cellular networks to initiate adaptive responses. Here, we demonstrate some new elements of this communication process, and we show that this networking is compromised by off-target, bioenergetic effects of some popular pharmacological tools. Treatment of cells with 5-aminoimidazole-4-carboxamide ribonucleoside (AICAR), so as to simulate elevated AMP levels, reduced the synthesis of bis-diphosphoinositol tetrakisphosphate ([PP] 2 -InsP 4 ), an intracellular signal that phosphorylates proteins in a kinase-independent reaction. This was a selective effect; levels of other inositol phosphates were unaffected by AICAR. By genetically manipulating cellular AMP-activated protein kinase activity, we showed that it did not mediate these effects of AICAR. Instead, we conclude that the simulation of deteriorating adenosine nucleotide balance itself inhibited [PP] 2 -InsP 4 synthesis. This conclusion is consistent with our demonstrating that oligomycin elevated cellular [AMP] and selectively inhibited [PP] 2 -InsP 4 synthesis without affecting other inositol phosphates. In addition, we report that the shortterm increases in [PP] 2 -InsP 4 levels normally seen during hyperosmotic stress were attenuated by 2-(2-chloro-4-iodophenylamino)-N-cyclopropylmethoxy-3,4-difluoro-benzamide (PD184352). The latter is typically considered an exquisitely specific mitogen-activated protein kinase kinase (MEK) inhibitor, but small interfering RNA against MEK or extracellular signal-regulated kinase revealed that this mitogen-activated protein kinase pathway was not involved. Instead, we demonstrate that [PP] 2 -InsP 4 synthesis was inhibited by PD184352 through its nonspecific effects on cellular energy balance. Two other MEK inhibitors, 1,4-diamino-2,3-dicyano-1,4-bis(methylthio)butadiene (U0126) and 2Ј-amino-3Ј-methoxyflavone (PD98059), had similar off-target effects. We conclude that the levels and hence the signaling strength of [PP] 2 -InsP 4 is supervised by cellular adenosine nucleotide balance, signifying a new link between signaling and bioenergetic networks.

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Section: Measurements Of Cellular Levels Of Inositol Phosphates and Amentioning

confidence: 99%

Cellular Energetic Status Supervises the Synthesis of Bis-Diphosphoinositol Tetrakisphosphate Independently of AMP-Activated Protein Kinase

Choi¹,

Mollapour²,

Choi³

et al. 2008

Mol Pharmacol

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“…The compounds were detected with a Perkin-Elmer LC-75 variable-wavelength detector, operating at 262 nm, combined with a Spectra Physics SP 4100 computing integrator. The first separation of the soluble nucleotides was done on a Whatman Partisil-10-SAX column (4 mm x 250 mm, particle size 1O ,tm) as described by Zakaria & Brown [15]. Separation was achieved with a phosphate buffer concentration gradient; the low-concentration buffer (A) was 5 mM-H3P04/KH2PO4, pH 4.0, and the high concentration buffer (B) was 0.5 M-KH2PO4/K2HPO4, pH 4.5.…”

Section: Chromatographic Proceduresmentioning

confidence: 99%

Pyrimidine metabolism and sugar nucleotide synthesis in rat liver

Rijcken

Hooghwinkel

Ferwerda

1990

Biochemical Journal

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With radioactive precursors, the labelling kinetics of the soluble pyrimidine nucleotides and of RNA were measured in rat liver to determine the contribution of the metabolic flows through synthesis de novo and the salvage pathway. To separate and quantify all pyrimidine nucleotides, an h.p.l.c. technique was developed using anion-exchange chromatography and reversed-phase chromatography. The concentrations of cytidine nucleotides were in the range of 30-45 nmol/g wet weight, and the concentrations of the uridine phosphates and of the UDP-sugars were approx. 6 and 20 times higher respectively. After a single injection of [14C]orotic acid and of [3H]cytidine, the specific radioactivities were determined as a function of time. The 1'C/3H ratio was calculated and gave a good indication of the involvement of the different flows. It could be concluded that UTP derived from synthesis de novo and from the salvage pathway is not completely mixed before being utilized. The flow of the salvage pathway is relatively more directed to RNA synthesis in the nucleus and that of synthesis de novo to cytoplasmic processes. For CTP it could also be concluded that the flow of the salvage pathway was relatively more directed to RNA synthesis in the nucleus. Because of the nuclear localization of the enzyme CMP-NeuAc (N-acetylneuraminate) synthase, special attention was paid to CMP-NeuAc. However, a conclusion about a location about the synthesis of CMP-NeuAc could not unequivocally be drawn, because of the small differences in 14C/3H ratio and the different values for the CDP-lipids.

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“…Opposite situation was observed in the case of 1-methyladenosine and 7-methylguanosine. pK b values for these compounds are, respectively, 7.6 and 7.1 [10,21]; below them both nucleosides are positively charged. An increase in the pH of NH 4 H 2 PO 4 is the reason for the decrease in load in the structure of nucleosides, and enlargement of retention as a consequence of the hydrophobic interaction formation.…”

Section: Apparatus and Chromatographic Conditionsmentioning

confidence: 93%

The influence of the mobile phase pH and the stationary phase type on the selectivity tuning in high performance liquid chromatography nucleosides separation

Kowalska

Krupczyńska

Buszewski³

2005

J of Separation Science

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Analysis of the modified nucleosides is particularly important in the medical area because of a possibility of cancerogenic processes studies. The aim of this work was to study the selectivity tuning of modified nucleosides through the investigations of interactions analyte (modified nucleoside) <==> stationary phase <==> mobile phase. A series of homemade stationary phases with different surface properties has been utilized. All of them contain various interaction sites such as: cholesterol (SG-CHOL); n-acylamide (SG-CHOL, SG-AP); aminopropyl (SG-CHOL, SG-AP, SG-NH2, SG-MIX); cyanopropyl, phenyl, octyl (SG-MIX), octadecyl (SG-MIX, SG-C18) and silanols localized on the silica gel surface of all packings. The attempt to predict the main interactions responsible for the retention between nucleosides and stationary phase ligands was done on the basis of the elemental analysis, and proportional part of an individual ligand bonded to silica surface results. In order to study the influence of different packing types on the analyzed nucleosides retention, the relationship between pH of the mobile phase buffer and the selectivity of a stationary phase was investigated.

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High-performance liquid column chromatography of nucleotides, nucleosides and bases

Cited by 164 publications

References 52 publications

Cellular Energetic Status Supervises the Synthesis of Bis-Diphosphoinositol Tetrakisphosphate Independently of AMP-Activated Protein Kinase

Cellular Energetic Status Supervises the Synthesis of Bis-Diphosphoinositol Tetrakisphosphate Independently of AMP-Activated Protein Kinase

Pyrimidine metabolism and sugar nucleotide synthesis in rat liver

The influence of the mobile phase pH and the stationary phase type on the selectivity tuning in high performance liquid chromatography nucleosides separation

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