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Published on: April 6, 2022
Quantification of extracellular UDP-galactose
1Cystic 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
Researchers developed a sensitive assay to detect UDP-galactose (UDP-Gal) release from cells. This finding suggests UDP-Gal acts as an important extracellular signaling molecule beyond its role in glycosylation.
Area of Science:
- Biochemistry
- Cell Biology
- Pharmacology
Background:
- The P2Y(14) receptor is activated by several UDP-sugars, including UDP-glucose (UDP-Glc) and UDP-N-acetylglucosamine (UDP-GlcNAc).
- While cellular release of UDP-Glc and UDP-GlcNAc is known, the endogenous agonists for the P2Y(14) receptor are not fully defined.
- UDP-galactose (UDP-Gal) is a potential endogenous agonist, but its extracellular presence and signaling role require further investigation.
Purpose of the Study:
- To develop a sensitive assay for quantifying UDP-Gal.
- To investigate the presence and release of UDP-Gal in various cell types.
- To determine if UDP-Gal functions as an extracellular signaling molecule.
Main Methods:
- Enzymatic conversion of UDP-Gal to UDP using 1-4-beta-galactosyltransferase.
- Phosphorylation of UDP to [gamma-(32)P]UTP using nucleoside diphosphokinase and [gamma-(32)P]ATP.
- High-performance liquid chromatography (HPLC) for monitoring [gamma-(32)P]UTP formation.
- Detection of UDP-Gal in cell cultures.
Main Results:
- A sensitive assay for UDP-Gal quantification was established, with linearity between 0.5 and 30 nM.
- Extracellular UDP-Gal was detected in resting cultures of multiple cell types.
- Stimulation of 1321N1 astrocytoma cells with thrombin led to increased UDP-Gal release.
Conclusions:
- UDP-Gal can be quantified with subnanomolar sensitivity using the developed assay.
- UDP-Gal is present extracellularly in various cell types.
- Regulated release of UDP-Gal suggests its role as a significant extracellular signaling molecule, in addition to its function in glycosylation.
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