鸡胚脑来源的(1→3)和(1→4)-N-乙酰-β-D-半乳糖胺基转移酶体外合成GbOse4Cer（红细胞糖苷脂）和GM2神经节苷脂。转移酶的溶解、纯化及特性鉴定。

In vitro biosynthesis of GbOse4Cer (globoside) and GM2 ganglioside by the (1-->3) and (1-->4)-N-acetyl beta-D-galactosaminyltransferases from embryonic chicken brain. Solubilization, purification, and characterization of the transferases.

作者信息

Schaeper R J, Das K K, Li Z, Basu S

机构信息

Department of Chemistry and Biochemistry, University of Notre Dame, Indiana 46556.

出版信息

Carbohydr Res. 1992 Dec 15;236:227-44. doi: 10.1016/0008-6215(92)85018-u.

DOI:10.1016/0008-6215(92)85018-u

PMID:1291049

Abstract

(1-->4)-N-Acetyl-beta-D-galactosaminyltransferase (GalNAcT-1) and (1-->3)-N-acetyl-beta-D-galactosaminyltransferase (GalNAcT-2), which are involved in the in vitro biosynthesis of GM2 and GbOse4Cer glycosphingolipids, respectively, have been solubilized and separated by differential detergent extraction from a membrane preparation of 19-day-old embryonic chicken brain. The separated GalNAcT-1 activity had a pH optima of 7.8-8.0, and the separated GalNAcT-2 activity a single pH optimum of 7.2. Furthermore, the partially purified GalNAcT-2 preparation catalyzed the transfer of N-acetylgalactosamine from UDP-D-[3H]GalNAc to only GbOse3Cer and nLcOse5Cer. Both GalNAcT-1 and GalNAcT-2 activities were purified to approximately 316- and 428-fold, respectively, by use of UDP-hexanolamine-Sepharose 4B affinity-column chromatography. However, the partially purified GalNAcT-1 preparation appeared to be active only with GM3, lactosylceramide, and lactotriaosylceramide. The proposed linkage of the N-acetylgalactosamine unit incorporated into GM3 is beta-D-GalpNAc-(1-->4)-GM3 from the isolation of [3H]threitol after hydrolysis of the desialylated, lead tetraacetate-treated, enzymic product, beta-D-GalpNAc-(1-->4)-beta-D-[6-3H]Galp-(1-->4)-beta-D-Glcp-(1-->1)-Cer . In addition, beta-D-GalpNAc-(1-->3)-GbOse3Cer was produced, as shown by the identification of 2,4,6-tri-O-methyl-galactose after permethylation and hydrolysis of the GalNAcT-2 enzymic product, GalpNAc-[6-3H]Galp--->Gal-->Glc-->Cer.

摘要

（1→4）-N-乙酰-β-D-半乳糖胺基转移酶（GalNAcT-1）和（1→3）-N-乙酰-β-D-半乳糖胺基转移酶（GalNAcT-2）分别参与GM2和GbOse4Cer神经节苷脂的体外生物合成，已通过不同的去污剂提取从19日龄鸡胚脑的膜制剂中溶解并分离出来。分离出的GalNAcT-1活性的最适pH为7.8 - 8.0，分离出的GalNAcT-2活性的最适pH为单一的7.2。此外，部分纯化的GalNAcT-2制剂催化N-乙酰半乳糖胺从UDP-D-[³H]GalNAc转移至仅GbOse3Cer和nLcOse5Cer。通过使用UDP-己醇胺-Sepharose 4B亲和柱色谱法，GalNAcT-1和GalNAcT-2的活性分别纯化至约316倍和428倍。然而，部分纯化的GalNAcT-1制剂似乎仅对GM3、乳糖基神经酰胺和乳糖三糖基神经酰胺有活性。从去唾液酸、经四乙酸铅处理的酶促产物β-D-GalpNAc-(1→4)-β-D-[6-³H]Galp-(1→4)-β-D-Glcp-(1→1)-Cer水解后分离出[³H]苏糖醇，推测GM3中掺入的N-乙酰半乳糖胺单元的连接方式为β-D-GalpNAc-(1→4)-GM3。此外，GalNAcT-2酶促产物GalpNAc-[6-³H]Galp--->Gal-->Glc-->Cer经全甲基化和水解后鉴定出2,4,6-三-O-甲基半乳糖，表明生成了β-D-GalpNAc-(1→3)-GbOse3Cer。