Comparison of extraction procedures for high-performance liquid chromatographic determination of cellular deoxynucleotides

Palmer, Sarah; Cox, Susan

doi:10.1016/0021-9673(94)89082-x

Cited by 22 publications

(27 citation statements)

References 10 publications

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“…Glycerol was omitted from the washing buffer, which did not affect ATP-binding characteristics. Deoxynucleotides were extracted from the filters by soaking them for 1 h in 700 mL 60% methanol (method adapted from Palmer and Cox, 1994). After incubation, 550 mL of the extracts were transferred to clean Eppendorf tubes and the liquid was evaporated using dry, flowing air.…”

Section: Nucleotide Extraction From Filtersmentioning

confidence: 99%

Mutations in the NB-ARC Domain of I-2 That Impair ATP Hydrolysis Cause Autoactivation

et al. 2006

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(M.A., T.L.) Resistance (R) proteins in plants confer specificity to the innate immune system. Most R proteins have a centrally located NB-ARC (nucleotide-binding adaptor shared by APAF-1, R proteins, and CED-4) domain. For two tomato (Lycopersicon esculentum) R proteins, I-2 and Mi-1, we have previously shown that this domain acts as an ATPase module that can hydrolyze ATP in vitro. To investigate the role of nucleotide binding and hydrolysis for the function of I-2 in planta, specific mutations were introduced in conserved motifs of the NB-ARC domain. Two mutations resulted in autoactivating proteins that induce a pathogen-independent hypersensitive response upon expression in planta. These mutant forms of I-2 were found to be impaired in ATP hydrolysis, but not in ATP binding, suggesting that the ATP-rather than the ADP-bound state of I-2 is the active form that triggers defense signaling. In addition, upon ADP binding, the protein displayed an increased affinity for ADP suggestive of a change of conformation. Based on these data, we propose that the NB-ARC domain of I-2, and likely of related R proteins, functions as a molecular switch whose state (on/off) depends on the nucleotide bound (ATP/ADP).

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Section: Nucleotide Extraction From Filtersmentioning

confidence: 99%

Mutations in the NB-ARC Domain of I-2 That Impair ATP Hydrolysis Cause Autoactivation

et al. 2006

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“…Prior to analysis by high-performance liquid chromatography (HPLC) the ribonucleotides in each sample were degraded by periodate oxidation (Tanaka et al, 1984;Palmer and Cox, 1994a). After periodate oxidation, all samples were spun at 2000 g for 20 min in a refrigerated centrifuge.…”

Section: Hplc Analysismentioning

confidence: 99%

“…Nucleotides were separated on a strong anion exchange column (Partisil 10 SAX, 250 x 4.6 mm J.D. ; Whatman, Clifton, NJ, USA) using a phosphate buffer gradient elution (Palmer and Cox, 1994a). The two buffers used were 0.02 M potassium phosphate (pH 3.5, buffer A) and 0.80 M potassium phosphate (pH 3.5, buffer B).…”

Section: Hplc Analysismentioning

confidence: 99%

Intracellular Metabolism of 3′-Azido-3′-Deoxythymidine in the Presence of Ganciclovir or Foscarnet

Palmer

Rasmussen

Harmenberg³

et al. 1996

Antivir Chem Chemother

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SummaryComparisons were made between the intracellular phosphorylation of 3'-azido-3'-deoxythymidine (AZT) alone and in combination with ganciclovir (GCV) or foscarnet (PFA) in lymphocytes, uninfected fibroblasts and CMV-infected fibroblasts. The effects of AZT and the combinations of AZT with GCV or PFA on cellular dNTP pools were also examined.The phosphorylation of AZT was not altered by the presence of GCV or PFA in lymphocytes, and neither AZT nor the combinations of AZT with GCV or PFA changed the levels of cellular dNTP pools in these cells. AZT was phosphorylated to a greater extent in lymphocytes when compared to fibroblasts, but the proportion of AZT di-and triphosphates was greater in fibroblasts.The infection of fibroblasts with CMV enhanced AZT phosphorylation and increased the levels of cellular dNTP pools. GCV caused a specific reduction in AZT phosphorylation in CMV-infected fibroblasts, with a seven-fold drop in AZT triphosphate compared to AZT alone. GCV did not affect AZT phosphorylation in uninfected fibroblasts, nor did GCV reduce the dTTP pool compared to AZT alone. The effects of GCV upon AZT phosphorylation in CMV-infected cells may shed light on the antagonistic effects of GCV upon the anti-HIV activity of AZT, and are of importance for the development of effective combination therapies for the treatment of AIDS patients infected with CMV.

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“…An analytical method for the quantitative measurement of NTP and dNTP pools in cells is therefore required for studying the influence of pharmacologically active agents on DNA and RNA synthesis and regulation. Both the separation techniques and the extraction procedure dramatically influence the efficiency of the analysis (1,2). In fact, determination of dNTP in cells and tissues is an analytical challenge since the concentration of dNTP present in mammalian cells is several orders of magnitude lower than the corresponding NTP (3), the latter being most likely to obscure the dNTP signals.…”

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confidence: 99%

“…Selective degradation of the cis-diol NTP by periodate and methylamine (3,4) has been proposed so as to oxidize NTP prior to high-performance liquid chromatography (HPLC) measurements of dNTP (5,6). Though this method is effective, some interfering products from the periodate oxidation coeluted with the dNTP fractions with MeOH but not TCA extraction (2). However, the periodate oxidation procedure was reported to result in partial destruction of dGTP (5).…”

mentioning

confidence: 99%

Simultaneous Determination of Deoxyribonucleoside in the Presence of Ribonucleoside Triphosphates in Human Carcinoma Cells by High-Performance Liquid Chromatography

Décosterd

Cottin

Chen

et al. 1999

Analytical Biochemistry

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Comparison of extraction procedures for high-performance liquid chromatographic determination of cellular deoxynucleotides

Cited by 22 publications

References 10 publications

Mutations in the NB-ARC Domain of I-2 That Impair ATP Hydrolysis Cause Autoactivation

Mutations in the NB-ARC Domain of I-2 That Impair ATP Hydrolysis Cause Autoactivation

Intracellular Metabolism of 3′-Azido-3′-Deoxythymidine in the Presence of Ganciclovir or Foscarnet

Simultaneous Determination of Deoxyribonucleoside in the Presence of Ribonucleoside Triphosphates in Human Carcinoma Cells by High-Performance Liquid Chromatography

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