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Anal Biochem. 2010 Jan 1;396(1):23-9. doi: 10.1016/j.ab.2009.08.022. Epub 2009 Aug 21.

Quantification of extracellular UDP-galactose.

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Cystic Fibrosis/Pulmonary Research and Treatment Center, Department of Medicine, 7017 Thurston-Bowles Building, CB 7248, University of North Carolina, Chapel Hill, NC 27599-7248, USA.


The human P2Y(14) receptor is potently activated by UDP-glucose (UDP-Glc), UDP-galactose (UDP-Gal), UDP-N-acetylglucosamine (UDP-GlcNAc), and UDP-glucuronic acid. Recently, cellular release of UDP-Glc and UDP-GlcNAc has been reported, but whether additional UDP-sugars are endogenous agonists for the P2Y(14) receptor remains poorly defined. In the present study, we describe an assay for the quantification of UDP-Gal with subnanomolar sensitivity. This assay is based on the enzymatic conversion of UDP-Gal to UDP, using 1-4-beta-galactosyltransferase. UDP is subsequently phosphorylated by nucleoside diphosphokinase in the presence of [gamma-(32)P]ATP and the formation of [gamma-(32)P]UTP is monitored by high-performance liquid chromatography. The overall conversion of UDP-Gal to [gamma-(32)P]UTP was linear between 0.5 and 30 nM UDP-Gal. Extracellular UDP-Gal was detected on resting cultures of various cell types, and increased release of UDP-Gal was observed in 1321N1 human astrocytoma cells stimulated with the protease-activated receptor agonist thrombin. The occurrence of regulated release of UDP-Gal suggests that, in addition to its role in glycosylation reactions, UDP-Gal is an important extracellular signaling molecule.

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