A method for the determination of N-acetylglucosaminyltransferase III activity in rat tissues involving HPLC

• Published in: Analytical biochemistry Vol. 170; no. 2; pp. 349 - 354
• Main Authors: Nishikawa, Atsushi, Fujii, Shigeru, Sugiyama, Toshihiro, Taniguchi, Naoyuki
• Format: Journal Article
• Language: English
• Published: San Diego, CA Elsevier Inc 01.05.1988; Elsevier
• Subjects: 2-aminopyridine; Analytical, structural and metabolic biochemistry; Animals; Biological and medical sciences; Brain - enzymology; Chromatography, High Pressure Liquid; Enzymes and enzyme inhibitors; Fundamental and applied biological sciences. Psychology; Glucosyltransferases - analysis; Glucosyltransferases - antagonists & inhibitors; HPLC; Hydrogen-Ion Concentration; Kidney - enzymology; Kinetics; Lung - enzymology; Magnesium - pharmacology; Magnetic Resonance Spectroscopy; Male; Myocardium - enzymology; N-acetylglucosaminyltransferase; N-Acetylglucosaminyltransferases; Rats; Spleen - enzymology; Testis - enzymology; Transferases; N-acetylglucosaminyltransferase; 2-aminopyridine; rats; HPLC
• ISSN: 0003-2697; 1096-0309
• DOI: 10.1016/0003-2697(88)90641-0

A fluorescence assay method for UDP-GlcNAc:glycopeptide β1-4 N-acetylglucosaminyltransferase (GnT-III) has been developed involving a pyridylaminated sugar as a substrate. A fluorescent sugar chain, in which the reducing end of the GlcNAcβ1-2Manα1-6(GlcNAcβ1-2Manα1-3)Manβ1-4GlcNAcβ1-4GlcNAc has been aminated with 2-aminopyridine, is incubated with an enzyme sample, and then the fluorescent product with a bisecting N-acetylglucosamine is separated by reverse-phase high performance liquid chromatography and quantitated with a fluorescence detector. This assay method was found to be sensitive enough for the detection of 0.1 pmol of a reaction product. This assay is a reliable alternative to the use of a radiolabeled substrate and can be used for assaying N-acetylglucosaminyltransferase activity in crude extracts of various rat tissues. The kinetic experiments were carried out using crude enzyme extracts from the rat kidney. The enzyme has a pH optimum of 6.25 and requires Mn 2+. The K m values for UDP-GlcNAc and a sugar acceptor substrate were found to be 3.1 m m and 190 μ m, respectively. The enzyme activity in the rat kidney was higher than those in the other tissues examined.