Thompson J N, Rodén L, Reynertson R
Anal Biochem. 1986 Feb 1;152(2):412-22. doi: 10.1016/0003-2697(86)90428-8.
The Sanfilippo A syndrome is characterized by a deficiency in heparin sulfamidase, which removes the N-sulfate groups of heparan sulfate and heparin in the course of normal catabolism of these polysaccharides. [N-35S]Heparin is the most commonly used substrate for the assay of sulfamidase activity but has certain disadvantages which have prompted us to search for alternative substrates. We report here on the use of heparin oligosaccharides for this purpose. The trisaccharide, GlcN-IdoUA-GlcN, and the pentasaccharide, GlcN-GlcUA-GlcN-GlcUA-GlcN, were N-sulfated with [35S]sulfur trioxide-trimethylamine complex; the tetrasaccharide, GlcN-UA-GlcN-UA, and the pentasaccharide, GlcN-IdoUA-GlcN-IdoUA-GlcN, were labeled by reduction with sodium borotritide followed by chemical N-sulfation. When incubated with sonicates of cultured skin fibroblasts from normal individuals, all four oligosaccharides were found to serve as substrates for heparin sulfamidase. Fibroblast sonicates from patients with the Sanfilippo A syndrome had little or no activity toward these substrates. Optimal activity of the enzyme was at pH 4.4-4.5. Comparison of the kinetic parameters showed that heparin had a lower Km than the oligosaccharides, whereas the Vmax values of the latter were higher than for heparin.
桑菲利波 A 综合征的特征是硫酸乙酰肝素酶缺乏，该酶在这些多糖的正常分解代谢过程中去除硫酸乙酰肝素和肝素的 N - 硫酸基团。[N - 35S]肝素是测定硫酸乙酰肝素酶活性最常用的底物，但有某些缺点，这促使我们寻找替代底物。我们在此报告肝素寡糖在此方面的应用。三糖 GlcN - IdoUA - GlcN 和五糖 GlcN - GlcUA - GlcN - GlcUA - GlcN 用 [35S]三氧化硫 - 三甲胺络合物进行 N - 硫酸化；四糖 GlcN - UA - GlcN - UA 和五糖 GlcN - IdoUA - GlcN - IdoUA - GlcN 先用硼氢化三钠还原然后进行化学 N - 硫酸化进行标记。当与正常个体培养的皮肤成纤维细胞的超声破碎物一起温育时，发现所有四种寡糖都可作为硫酸乙酰肝素酶的底物。桑菲利波 A 综合征患者的成纤维细胞超声破碎物对这些底物几乎没有或没有活性。该酶的最佳活性在 pH 4.4 - 4.5。动力学参数比较表明，肝素的 Km 比寡糖低，而后者的 Vmax 值比肝素高。
