A method for the determination of N-acetylglucosaminyltransferase III activity in rat tissues involving HPLC.
A fluorescence assay method for UDP-GlcNAc:glycopeptide beta 1-4 N-acetylglucosaminyltransferase (Gn T-III) has been developed involving a pyridylaminated sugar as a substrate. A fluorescent sugar chain, in which the reducing end of the GlcNAc beta 1-2Man alpha 1-6(GlcNAc beta 1-2Man alpha 1-3)Man beta 1-4GlcNAc beta 1-4GlcNAc 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 Mn2+. The Km values for UDP-GlcNAc and a sugar acceptor substrate were found to be 3.1 mM and 190 microM, respectively. The enzyme activity in the rat kidney was higher than those in the other tissues examined.
Nishikawa
A
A
Department of Biochemistry, Osaka University Medical School, Japan.
Fujii
S
S
Sugiyama
T
T
Taniguchi
N
N
D000818:Animals; D001921:Brain / Q000201:enzymology; D002851:Chromatography, High Pressure Liquid; D005964:Glucosyltransferases / Q000032:analysis* / Q000037:antagonists & inhibitors; D006863:Hydrogen-Ion Concentration; D007668:Kidney / Q000201:enzymology; D007700:Kinetics; D008168:Lung / Q000201:enzymology; D008274:Magnesium / Q000494:pharmacology; D009682:Magnetic Resonance Spectroscopy; D008297:Male; D009206:Myocardium / Q000201:enzymology; D017351:N-Acetylglucosaminyltransferases; D051381:Rats; D013154:Spleen / Q000201:enzymology; D013737:Testis / Q000201:enzymology
D005964:Glucosyltransferases; D017351:N-Acetylglucosaminyltransferases; C054599:beta-1,4-mannosyl-glycoprotein beta-1,4-N-acetylglucosaminyltransferase; D008274:Magnesium