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Journal ArticleDOI

Enzymic determination of uracil nucleotides in tissues.

01 Nov 1970-Analytical Biochemistry (Academic Press)-Vol. 38, Iss: 1, pp 105-114
TL;DR: An enzymic assay for the sequential determination of UDP-glucose, UDP-galactose, UTP, UDP, and 5′-UMP in tissues is described, which uses commercially available enzymes and is highly reproducible, with standard deviations of less than 2%.
About: This article is published in Analytical Biochemistry.The article was published on 1970-11-01. It has received 123 citations till now. The article focuses on the topics: Uracil nucleotide & Uracil.
Citations
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Journal ArticleDOI
TL;DR: Results directly demonstrate the mechanically induced release of UTP and illustrate the efficient coupling of this release to activation of P2Y4 receptors.

298 citations

Journal ArticleDOI
TL;DR: Depression of the concentration of UTP as substrate for RNA polymerases and its reversal by uridine provides a new means to inhibit RNA synthesis in vivo for defined time periods.

226 citations

Journal ArticleDOI
TL;DR: The evidence that pyrimidine nucleotides exert their effects by binding to distinct pyrimidinoceptors, which are coupled to pertussis toxin-sensitive G proteins in human phagocytes is reviewed.

177 citations

Journal ArticleDOI
TL;DR: Detecting 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.
Abstract: The wide distribution of the uridine nucleotide-activated P2Y2, P2Y4 and P2Y6 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 [14C]-glucose-1P by the enzyme UDP-glucose pyrophosphorylase which allows quantification of UTP in the sub-nanomolar concentration range. The UTP-dependent conversion of [14C]-glucose-1P to [14C]-UDP-glucose was made irreversible by including the pyrophosphate scavenger inorganic pyrophosphatase in the reaction medium and [14C]-glucose-1P and [14C]-UDP-glucose were separated and quantified by HPLC. Formation of [14C]-UDP-glucose was linearly observed between 1 and 300 nM UTP. The reaction was highly specific for UTP and was unaffected by a 1000 fold molar excess of ATP over UTP. 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 cm2 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. 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.

151 citations


Cites methods from "Enzymic determination of uracil nuc..."

  • ...The sensitivity of this coupled enzymatic assay using [14C]-glucose as a radiolabelled cosubstrate is at least two orders of magnitude greater than other methods previously available (Keppler et al., 1970 ; SasvariSzekely et al., 1975)....

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References
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Journal ArticleDOI
01 Jul 1960
TL;DR: In this article, a pair of tongs are used to compress large tissue pieces and organs of animals to a thin layer and can thus be frozen in a fraction of a second.
Abstract: 1. A pair of tongs was designed the mouth of which consists of two blocks of a metal of high thermal conductivity (aluminum). Prior to use the metal is cooled in liquid air or nitrogen. With this tool large tissue pieces and whole organs of animals can be compressed in situ to a thin layer and can thus be frozen in a fraction of a second. The fall in temperature was measured with a thermocouple and was recorded with a loop oscillograph. Measurements of the cooling velocity in liquid air and in isopentane of −150°C were carried out for comparison. 2. The cooling process in the compressed tissue was analyzed with the aid of the theory of heat conduction, and the heat conduction equation holding for the present system was derived. It yields values for velocity of cooling which agree well with the experimental data in the temperature range 38°2-0°C. The cause of the deviation at lower temperatures is discussed. 3. Instructions are given for the estimation of the time required to cool down the central layer of the tissue held in the tongs to any specified temperature.

827 citations

Journal ArticleDOI
TL;DR: Repeated intraperitoneal injections of d-galactosamine produce within 24–48 hours in rat livers alterations that closely resemble human viral hepatitis, indicating a high specificity of d -galactoamine.

502 citations

Journal ArticleDOI
TL;DR: It is concluded that the compound found with treatment of UDPG with an enzyme of S. fragilis is uridine diphosphate galactose, and the bearing of this finding on the mechanism of action of UDPG is discussed.

386 citations

Journal ArticleDOI
TL;DR: In the initial studies (5) it was found that a thermostable factor was required in the over-all Reactions II and III, which led eventually to the discovery and isolation of glucose diphosphate, which acts as a coenzyme in Reaction III.

318 citations