Sakamoto Y, Taguchi T, Tano Y, Ogawa T, Leppänen A, Kinnunen M, Aitio O, Parmanne P, Renkonen O, Taniguchi N
Department, Osaka University Medical School, Suita, Osaka 565-0871, Japan.
J Biol Chem. 1998 Oct 16;273(42):27625-32. doi: 10.1074/jbc.273.42.27625.
A beta1,6N-acetylglucosaminyltransferase (beta1-6GnT) responsible for the formation of the beta1,6-branched poly-N-acetyllactosamine structure has been purified 210,000-fold in 2.4% yield from a homogenate of hog small intestine by successive column chromatographies involving CM-Sepharose FF, Ni2+-chelating Sepharose FF, and UDP-hexanolamine-agarose, using an assay wherein pyridylaminated lacto-N-neotetraose (Galbeta1-4GlcNAcbeta1-3Galbeta1-4Glc-PA) was used as an acceptor substrate, and the reaction product was Galbeta1-4GlcNAcbeta1-3(GlcNAcbeta1-6)Galbeta1-4 Glc-PA. The apparent molecular weight of the purified enzyme was 76,000 under nonreducing conditions. The enzyme has a pH optimum at 7.0 and has no requirement for any divalent metal ions. The Km values for pyridylaminated lacto-N-neotetraose and UDP-GlcNAc were 0.96 and 2. 59 mM, respectively. For its activity, this enzyme was shown to have an absolute requirement of at least a complete LacNAc (LacNAc = Galbeta1-4GlcNAc) residue bound to position 3 of the acceptor Gal residues, i.e. it is capable of acting only on the Gal residues of internal LacNAc units. The data strongly suggest that this enzyme could be involved in generating branches to central positions of preformed as well as growing polylactosamine chains, but not in synthesizing the distal branches to growing polylactosamine chains.
一种负责形成β1,6-分支的聚N-乙酰乳糖胺结构的β1,6-N-乙酰葡糖胺基转移酶（β1-6GnT），通过连续的柱色谱法，包括CM-琼脂糖凝胶FF、Ni2+螯合琼脂糖凝胶FF和UDP-己醇胺琼脂糖，从猪小肠匀浆中以2.4%的产率纯化了210,000倍，使用的测定方法是将吡啶基化的乳糖-N-新四糖（Galβ1-4GlcNAcβ1-3Galβ1-4Glc-PA）用作受体底物，反应产物为Galβ1-4GlcNAcβ1-3（GlcNAcβ1-6）Galβ1-4Glc-PA。在非还原条件下，纯化酶的表观分子量为76,000。该酶的最适pH为7.0，不需要任何二价金属离子。吡啶基化的乳糖-N-新四糖和UDP-GlcNAc的Km值分别为0.96和2.59 mM。就其活性而言，该酶显示出绝对需要至少一个完整的乳糖胺（LacNAc = Galβ1-4GlcNAc）残基与受体Gal残基的3位结合，即它仅能作用于内部乳糖胺单元的Gal残基。数据强烈表明，这种酶可能参与在预先形成的以及正在生长的聚乳糖胺链的中心位置产生分支，但不参与合成正在生长的聚乳糖胺链的远端分支。
