Preparation of a high-affinity photolabeling reagent for the Gal/GalNAc lectin of mammalian liver: demonstration of galactose-combining sites on each subunit of rabbit hepatic lectin.
Preparation of a high-affinity photolabeling reagent for the Gal/GalNAc lectin of mammalian liver: demonstration of galactose-combining sites on each subunit of rabbit hepatic lectin.
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DOI：
10.1021/bi00370a016
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发表时间：
1986-11
期刊：
影响因子：
2.9
通讯作者：
R. Lee;Y. Lee
中科院分区：
文献类型：
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作者：
R. Lee;Y. Lee
基于兔肝的D-半乳糖/N-乙酰基-D-半乳糖胺特异性凝集素可以耐受半乳糖苷的C-6羟基上的大基团的知识[Lee，R. T.（1982）Biochemistry 21，1045-1050]，我们从去唾液酸胎球蛋白的三触角糖肽级分制备了用于该凝集素的高亲和力光标记试剂。在温和条件下，D-吡喃半乳糖苷的C-6羟基被转化为伯氨基。该方法包括用半乳糖氧化酶将羟基转化为氧代基团，然后用苄胺和氰基硼氢化钠进行还原胺化。苄基氨基衍生物的催化氢解产生所需的6-氨基-6-脱氧-D-半乳糖苷。将4-叠氮基苯甲酰基连接到新产生的氨基上以产生可光活化的亲和标记试剂。该试剂以光依赖性和亲和力依赖性的方式标记Triton-solubilized，纯化的兔和大鼠肝凝集素。凝集素的所有多肽亚基都被标记，表明每个亚基含有至少一个D-半乳糖结合位点。在兔肝凝集素的情况下，次要亚基（46 kDa）的标记比主要的（40 kDa）更有效。
On the basis of the knowledge that the D-galactose/N-acetyl-D-galactosamine-specific lectin of rabbit liver can tolerate a large group on the C-6 hydroxyl group of a galactoside [Lee, R. T. (1982) Biochemistry 21, 1045-1050], we prepared a high-affinity photolabeling reagent for this lectin from a triantennary glycopeptide fraction of asialofetuin. The C-6 hydroxyl group of a D-galactopyranoside was converted, under mild conditions, into a primary amino group. The procedure involves conversion of the hydroxyl group to an oxo group with galactose oxidase, followed by reductive amination using benzylamine and sodium cyanoborohydride. Catalytic hydrogenolysis of the benzylamino derivative yielded the desired 6-amino-6-deoxy-D-galactoside. A 4-azidobenzoyl group was attached to the newly produced amino group to yield a photoactivatable affinity-labeling reagent. The reagent labeled the Triton-solubilized, purified hepatic lectins of rabbit and rat in a photo- and affinity-dependent manner. All the polypeptide subunits of the lectins were labeled, indicating that each subunit contains at least one D-galactose-combining site. In the case of the rabbit hepatic lectin, the minor subunit (46 kDa) was labeled more efficiently than the major one (40 kDa).