Determination of UTP and ATP pool sizes in human tonsillar lymphocytes by using Escherichia coli RNA polymerase

Sasvári-Székely, Mária; Vitéz, M.; Staub, Maria; Antoni, F

doi:10.1016/0005-2787(75)90192-6

Cited by 17 publications

(4 citation statements)

References 8 publications

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“…The assay for UTP described here provides reliable detection of UTP for the first time in biological samples such as cell supernatants and cell lysates. The sensitivity of this coupled enzymatic assay using [ 14 C]‐glucose as a radiolabelled co‐substrate is at least two orders of magnitude greater than other methods previously available ( Keppler et al ., 1970 ; Sasvari‐Szekely et al ., 1975 ). Moreover, we show here using radiolabelled UTP in proof of principle experiments that the assay is linear to at least the low picomolar range of concentrations of UTP.…”

Section: Discussionmentioning

confidence: 99%

Quantitation of extracellular UTP using a sensitive enzymatic assay

Lazarowski

Harden

1999

British J Pharmacology

139

135

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1 The wide distribution of the uridine nucleotide-activated P2Y 2 , P2Y 4 and P2Y 6 receptors suggests a role for UTP as an important extracellular signalling molecule. However, direct evidence for UTP release and extracellular accumulation has been addressed only recently due to the lack of a sensitive assay for UTP mass. In the present study, we describe a method that is based on the uridinylation of [ ]-UDP-glucose was linearly observed between 1 and 300 nM UTP. The reaction was highly speci®c for UTP and was unaected by a 1000 fold molar excess of ATP over UTP. 3 Release of UTP was measured with a variety of cells including platelets and leukocytes, primary airway epithelial cells, rat astrocytes and several cell lines. In most resting attached cultures, extracellular UTP concentrations were found in the low nanomolar range (1 ± 10 nM in 0.5 ml medium bathing 2.5 cm 2 dish). Up to a 20 fold increase in extracellular UTP levels was observed in cells subjected to a medium change. Extracellular UTP levels were 10 ± 30% of the ATP levels in both resting and mechanically-stimulated cultured cells. In unstirred platelets, a 1 : 100 ratio UTP/ ATP was observed. Extracellular UTP and ATP increased 10 fold in thrombin-stimulated platelets. 4 Detection of UTP in nanomolar concentrations in the medium bathing resting cultures suggests that constitutive release of UTP may provide a mechanism of regulation of the basal activity of uridine nucleotide sensitive receptors.

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Section: Discussionmentioning

confidence: 99%

Quantitation of extracellular UTP using a sensitive enzymatic assay

Lazarowski

Harden

1999

British J Pharmacology

139

135

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“…After 12 h of incubation at 40~ toluene-based scintillation fluid was added and the radioactivity was determined in a liquid scintillation counter (Betaszint 5000; Berthold-Frieseke, Karlsruhe, Federal Republic of Germany). (b) Determination of the endogenous UTP pool sizes of the different oocyte classes was carried out by a modification of the very sensitive method described by Sasv~tri-Sz~kely et al (70). Oocytes without the surrounding epithelium were homogenized in 0.5 N PCA at 4~ as described above (10 SCHEER, TRENDELENBURG, AND FRANKE Transcription of Genes of rRNA in Oogenesis 467 mature and 10 vitellogenic oocytes each in 1 ml, 100 previtellogenic oocytes each in 0.2 ml of PCA).…”

Section: Analysis Of the Nucleotide Poolmentioning

confidence: 99%

Regulation of transcription of genes of ribosomal rna during amphibian oogenesis. A biochemical and morphological study.

Scheer¹,

Trendelenburg²,

Franke³

1976

The Journal of Cell Biology

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Natural changes in the transcription of rRNA genes were studied in nucleoli from three oogenic stages of the newt Triturus alpestris with electron microscope, autoradiographic, and biochemical techniques. From determinations of the uridine triphosphate pool sizes and [SH]uridine uptake, phosphorylation, and incorporation into 28S and 18S rRNAs in vivo it was estimated that the rate of rRNA synthesis was about 0.01% in previtellogenic oocytes and 13% in mature oocytes when compared to midvitellogenesis. Spread preparations of nucleoli showed significant morphological changes in the transcriptional complexes. The total number of lateral fibrils, i.e., ribonucleoproteins containing the nascent rRNA precursor, were drastically decreased in stages of reduced synthetic activity. This indicates that rRNA synthesis is regulated primarily at the level of transcription. The resulting patterns of fibril coverage of the nucleolar chromatin axes revealed a marked heterogeneity. On the same nucleolar axis occurred matrix units that were completely devoid of lateral fibrils, matrix units that were almost fully covered with lateral fibrils, and various forms of matrix units with a range of lateral fibril densities intermediate between the two extremes. Granular particles that were tentatively identified as RNA polymerase molecules were not restricted to the transcriptional complexes. They were observed, although less regularly and separated by greater distances, in untranscribed spacer regions as well as in untranscribed gene intercepts. The results show that the pattern of transcriptional control of rRNA genes differs widely in different genes, even in the same genetic unit.

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“…Determination of the Specific Activity of the ATP Pool-The method of Lee et al (33), based on the work of Sasvá ri-Székely et al (34), was used to determine the specific activity of the ATP pool of HeLa cells. Briefly, the technique makes use of the fact that the number of AMP residues equals the number of UMP residues when RNA is polymerized using poly(dA-dT) as the template.…”

Section: Cellsmentioning

confidence: 99%

Turnover of the Acyl Phosphates of Human and Murine Prothymosin α in Vivo

Wang

Tao

Trumbore

et al. 1997

Journal of Biological Chemistry

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Prothymosin ␣ is a small, highly acidic, abundant, nuclear, mammalian protein which is essential for cell growth. Our laboratory has recently shown that primate prothymosin ␣ contains stoichiometric amounts of phosphate on the glutamyl groups of the protein and that in vitro the phosphate undergoes rapid hydrolysis or transfer to a nearby serine residue. Here an assay for the presence of acyl phosphates in vivo has been developed by measuring stable phosphoserine and phosphothreonine in vitro. The assay was used to determine the half-life of the acyl phosphates on prothymosin ␣ in vivo by pulse-labeling HeLa cells with [ 32 P]orthophosphate and chasing using three different techniques: permeabilization with digitonin to allow extracellular ATP to equilibrate with the intracellular pool; electroporation in the presence of ATP to reduce the specific activity of Prothymosin ␣ is a small, highly acidic (1, 2), nuclear (3-6) protein found in virtually all mammalian tissues (7-11). Under conditions of rapid growth, a cultured cell accumulates upwards of 17 million molecules, a number roughly equivalent to that of histone cores (12). When cells are disrupted with detergents, the protein readily leaks out of the nucleus, suggesting that stable interactions with nucleosomes or with the nuclear matrix are not an inherent part of its activity (2, 3). Its precise function is unknown. Nevertheless, a role in cell proliferation has been proposed based on the following observations: prothymosin ␣ mRNA is plentiful only in rapidly dividing cells (13)(14)(15); the level of the protein declines 10-fold in cells forced to subsist in stationary phase (12); the amount of prothymosin ␣ is directly proportional to the proliferative activity of the tissue from which it is isolated (13); and the uptake of antisense oligodeoxyribonucleotides directed toward various locations in prothymosin ␣ mRNA prevents synchronized human myeloma cells from entering mitosis (16). Hence, a deficiency in prothymosin ␣ is associated with failure to complete the cell cycle.Prothymosin ␣ has several unusual features. The human protein, which is almost identical to that of all other mammals (17), has 109 amino acids, nearly 50% of which are acidic (1, 2). A potent nuclear localization signal consisting of five basic amino acids has been identified near the carboxyl terminus (3, 6), while a second cluster of five basic residues near the amino terminus has no unambiguous role (3,18). The absence of all aromatic residues renders the protein transparent at 280 nm; it also lacks methionine, cysteine, and histidine. Based on biophysical data, it is believed to have an unfolded structure (19), a conclusion consistent with the presence of only seven widely dispersed hydrophobic residues in the human protein. Prothymosin ␣ does not bind SDS and stains anomalously with silver stain (2). The protein and the peptides derived from it exhibit poor immunogenicity. However, due to yet another aberrant property, the ability to partition quantitatively into the aqueous phase ...

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Determination of UTP and ATP pool sizes in human tonsillar lymphocytes by using Escherichia coli RNA polymerase

Cited by 17 publications

References 8 publications

Quantitation of extracellular UTP using a sensitive enzymatic assay

Quantitation of extracellular UTP using a sensitive enzymatic assay

Regulation of transcription of genes of ribosomal rna during amphibian oogenesis. A biochemical and morphological study.

Turnover of the Acyl Phosphates of Human and Murine Prothymosin α in Vivo

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