Nishikawa A, Fujii S, Sugiyama T, Taniguchi N
Department of Biochemistry, Osaka University Medical School, Japan.
Anal Biochem. 1988 May 1;170(2):349-54. doi: 10.1016/0003-2697(88)90641-0.
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.
已开发出一种用于UDP - GlcNAc：糖肽β1 - 4 N - 乙酰葡糖胺基转移酶（GnT - III）的荧光测定方法，该方法使用吡啶基化糖作为底物。将还原端为GlcNAcβ1 - 2Manα1 - 6(GlcNAcβ1 - 2Manα1 - 3)Manβ1 - 4GlcNAcβ1 - 4GlcNAc且已用2 - 氨基吡啶胺化的荧光糖链与酶样品一起孵育，然后通过反相高效液相色谱法分离出具有平分型N - 乙酰葡糖胺的荧光产物，并用荧光检测器进行定量。发现该测定方法对检测0.1 pmol的反应产物足够灵敏。该测定是使用放射性标记底物的可靠替代方法，可用于测定各种大鼠组织粗提物中的N - 乙酰葡糖胺基转移酶活性。动力学实验使用大鼠肾脏的粗酶提取物进行。该酶的最适pH为6.25，需要Mn2 +。发现UDP - GlcNAc和糖受体底物的Km值分别为3.1 mM和190 μM。大鼠肾脏中的酶活性高于所检查的其他组织中的酶活性。
