title: Quantification of extracellular UDP-galactose.
abstract: 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.
authors.affiliation: 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. eduardo_lazarowski@med.unc.edu
authors.forename: Eduardo R
authors.initials: ER
authors.lastname: Lazarowski
mesh_terms: D000818:Animals; D055162:Biocatalysis / Q000187:drug effects; D002417:Cattle; D002460:Cell Line; D004285:Dogs; D005110:Extracellular Space / Q000737:chemistry* / Q000187:drug effects; D005700:Galactosyltransferases / Q000378:metabolism; D006801:Humans; D007785:Lactose / Q000096:biosynthesis; D009701:Nucleoside-Diphosphate Kinase / Q000378:metabolism; D010766:Phosphorylation / Q000187:drug effects; D013917:Thrombin / Q000494:pharmacology; D014531:Uridine Diphosphate Galactose / Q000032:analysis* / Q000378:metabolism*; D014532:Uridine Diphosphate Glucose / Q000378:metabolism
chemical_list: D014531:Uridine Diphosphate Galactose; D005700:Galactosyltransferases; D009701:Nucleoside-Diphosphate Kinase; D013917:Thrombin; D007785:Lactose; D014532:Uridine Diphosphate Glucose
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