Gu J, Nishikawa A, Fujii S, Gasa S, Taniguchi N
Department of Biochemistry, Osaka University Medical School, Japan.
J Biol Chem. 1992 Feb 15;267(5):2994-9.
The beta-galactoside beta 1-6- and beta 1-3-N-acetylglucosaminyltransferases (beta 1-6GnT and beta 1-3GnT) that synthesize blood group I and i antigens were identified in rat tissues, using pyridylaminated lacto-N-neotetraose (Gal beta 1-4GlcNAc beta 1-3Gal beta 1-4Glc-PA) as an acceptor. The products of the transferase reactions were separated on high performance liquid chromatography. The products of the transferase reactions were identified by 1H NMR as (formula; see text) and GlcNAc beta 1-3Gal beta 1-4GlcNAc beta 1-3Gal beta 1-4Glc-PA. The product for beta 1-6GnT was also identified by methylation analysis. Kinetic experiments were carried out using rat serum for beta 1-3GnT and partially purified enzyme from rat intestine for beta 1-6GnT. beta 1-3GnT has a pH optimum of 7.5 and requires Mn2+ for optimal activity. beta 1-6GnT has a pH optimum of 7.0 and does not require Mn2+. Studies on the substrate specificity of each enzyme indicated that the preferred substrate for beta 1-3GnT had the general structure Gal beta 1-4GlcNAc-OR and, for beta 1-6GnT, Gal beta 1-4GlcNAc beta 1-3Gal-OR where R = sugar. This is the first demonstration that the beta 1-6GnT acts on an internal galactose of lacto-N-neotetraose and paragloboside, and the enzyme appears to be a novel enzyme in terms of substrate specificity.
利用吡啶胺化的乳糖-N-新四糖（Galβ1-4GlcNAcβ1-3Galβ1-4Glc-PA）作为受体，在大鼠组织中鉴定出了合成血型I和i抗原的β-半乳糖苷β1-6-和β1-3-N-乙酰葡糖胺基转移酶（β1-6GnT和β1-3GnT）。转移酶反应的产物通过高效液相色谱进行分离。转移酶反应的产物经1H NMR鉴定为（分子式；见正文）和GlcNAcβ1-3Galβ1-4GlcNAcβ1-3Galβ1-4Glc-PA。β1-6GnT的产物也通过甲基化分析进行了鉴定。使用大鼠血清进行β1-3GnT的动力学实验，使用大鼠肠道的部分纯化酶进行β1-6GnT的动力学实验。β1-3GnT的最适pH为7.5，最佳活性需要Mn2+。β1-6GnT的最适pH为7.0，不需要Mn2+。对每种酶的底物特异性研究表明，β1-3GnT的优选底物具有通用结构Galβ1-4GlcNAc-OR，而β1-6GnT的优选底物为Galβ1-4GlcNAcβ1-3Gal-OR，其中R = 糖。这是首次证明β1-6GnT作用于乳糖-N-新四糖和副球蛋白的内部半乳糖，并且就底物特异性而言，该酶似乎是一种新型酶。
