title: Specific Detection of Cellular Glutamine Hydrolysis in Live Cells Using HNCO Triple Resonance NMR.
abstract: Glutamine plays key roles as a biosynthetic precursor or an energy source in cancers, and interest in its metabolism is rapidly growing. However, the proper evaluation of glutamine hydrolysis, the very first reaction in the entire glutaminolysis, has been difficult. Here, we report a triple resonance NMR-based assay for specific detection of glutaminase activity carrying out this reaction using stable-isotope labeled glutamine. Compared to conventional methods involving coupled enzyme assays, the proposed approach is direct because it detects the presence of the H-N-CO amide spin system. In addition, the method is unique in enabling the measurement of glutamine hydrolysis reaction in real-time in live cells. The approach was applied to investigating the effects of a glutaminase inhibitor and the inhibitory effects of glucose on glutamine metabolism in live cells. It can be easily applied to studying other signals that affect cellular glutamine metabolism.
authors.affiliation: Natural Product Research Institute, College of Pharmacy, Seoul National University , Sillim-dong, Gwanak-gu, Seoul, 151-742, Korea.
authors.forename: Sujin
authors.initials: S
authors.lastname: Lee
authors.affiliation: Natural Product Research Institute, College of Pharmacy, Seoul National University , Sillim-dong, Gwanak-gu, Seoul, 151-742, Korea.|Department of Biochemistry and Molecular Biology, School of Medicine, Shenzhen University , Shenzhen, 518060, China.
authors.forename: He
authors.initials: H
authors.lastname: Wen
authors.affiliation: Natural Product Research Institute, College of Pharmacy, Seoul National University , Sillim-dong, Gwanak-gu, Seoul, 151-742, Korea.|Natural Constituents Research Center, Korea Institute of Science and Technology (KIST) , Gangneung, 25451, Korea.
authors.forename: Jin Wook
authors.initials: JW
authors.lastname: Cha
authors.affiliation: Natural Product Research Institute, College of Pharmacy, Seoul National University , Sillim-dong, Gwanak-gu, Seoul, 151-742, Korea.
authors.forename: Sunghyouk
authors.initials: S
authors.lastname: Park
mesh_terms: D000818:Animals; D045744:Cell Line, Tumor; D005947:Glucose / Q000378:metabolism; D005973:Glutamine / Q000378:metabolism*; D006868:Hydrolysis; D051379:Mice; D019906:Nuclear Magnetic Resonance, Biomolecular / Q000379:methods*
chemical_list: D005973:Glutamine; D005947:Glucose