Hopwood J J, Elliott H
Clin Chim Acta. 1981 Apr 27;112(1):55-66. doi: 10.1016/0009-8981(81)90268-0.
(1) A series of tritiated oligosaccharides, 2-sulfamino-2-deoxy-D-[1-14C]glucose (GlcNS) and [sulfamino-34S]heparin were evaluated as substrates for sulfamidase present in cultured human skin fibroblasts. (2) The following radiolabelled disaccharides were prepared from heparin: O-(alpha-2-sulfamino-2-deoxy-D-glucopyranosyl)-(1 leads to 3)-L-[6,3H]idonic acid (GlcNS-IdOA) and O-(alpha-2-sulfamino-2-deoxy-D-glucopyranosyl)-(1 leads to 3)-2,5 anhydro-L-[6,3H]idonic acid (HlcNS-anIdOA). Other radiolabelled oligosaccharides evaluated as sulfamidase substrates were the disaccharides O-(alpha-2-sulfamino-2-deoxy-D-glucopyranosyl)-(1 leads to 4)-L-[6,3H]idose (GlcNS-Ido) and O-(alpha-2-sulfamino-2-deoxy-D-glucopyranosyl)-)1 leads to 4)-L-[6,3H]idose 2-sulfate (GlcNS-Ido(OS)) and a preparation containing the tetrasaccharide GlcNS-UA-GlcNS-l-idonic acid, GlcNS-UA-GlcNS-anhydro-L-idonic acid and GlcNS-UA-GlcNS-L-gulonic acid. (3) Sulfamidase activity assessed with GlcNS-IdOA and GlcNS-anIdOA were approximately equal and up to 4, 8 and 800 times higher than the value obtained using [sulfamino-35S]heparin, GlcNS-Ido(OS) and GlcNS-Ido respectively. Under the assay conditions used GlcNS was not de-N-sulfated. These results demonstrate that C6 carboxyl and C2 sulfate ester groups on the adjacent residue to the sulfaminoglucosamine moiety are important structural requirements in the mechanism of action or binding of sulfamidase toward N-sulfated disaccharides. The results obtained for a partially characterized mixture of tetrasaccharides suggest that they are degraded four times faster than their disaccharide structural counterparts. (4) No detectable sulfamidase activity toward [sulfamino-35S]heparin, monosaccharide, disaccharide or tetrasaccharide substrates could be detected using homogenates of fibroblast cultures from Sanfilippo A patients (sulfamidase deficient). (5)sulfamidase activity measured with GlcNS-IdOA exhibited a pH optimum at 4.5 to 5.5, an apparent Km of approximately 220 mumol/l and potent inhibition by sulfate ions.
(1) 一系列氚标记的寡糖、2-氨基磺酰基-2-脱氧-D-[1-¹⁴C]葡萄糖（GlcNS）和[氨基磺酰基-³⁴S]肝素被评估为培养的人皮肤成纤维细胞中存在的氨磺酰酶的底物。(2) 从肝素制备了以下放射性标记的二糖：O-(α-2-氨基磺酰基-2-脱氧-D-吡喃葡萄糖基)-(1→3)-L-[6,³H]艾杜糖醛酸（GlcNS-IdOA）和O-(α-2-氨基磺酰基-2-脱氧-D-吡喃葡萄糖基)-(1→3)-2,5-脱水-L-[6,³H]艾杜糖醛酸（HlcNS-anIdOA）。作为氨磺酰酶底物评估的其他放射性标记寡糖是二糖O-(α-2-氨基磺酰基-2-脱氧-D-吡喃葡萄糖基)-(1→4)-L-[6,³H]艾杜糖（GlcNS-Ido）和O-(α-2-氨基磺酰基-2-脱氧-D-吡喃葡萄糖基)-(1→4)-L-[6,³H]艾杜糖2-硫酸酯（GlcNS-Ido(OS)）以及一种含有四糖GlcNS-UA-GlcNS-L-艾杜糖醛酸、GlcNS-UA-GlcNS-脱水-L-艾杜糖醛酸和GlcNS-UA-GlcNS-L-古洛糖酸的制剂。(3) 用GlcNS-IdOA和GlcNS-anIdOA评估的氨磺酰酶活性大致相等，分别比使用[氨基磺酰基-³⁵S]肝素、GlcNS-Ido(OS)和GlcNS获得的值高4倍、8倍和80倍。在所使用的测定条件下，GlcNS未被脱N-磺化。这些结果表明，氨磺酰葡糖胺部分相邻残基上的C6羧基和C2硫酸酯基团是氨磺酰酶对N-硫酸化二糖作用机制或结合的重要结构要求。对于部分表征的四糖混合物获得的结果表明，它们的降解速度比其二糖结构对应物快四倍。(4) 使用Sanfilippo A患者（氨磺酰酶缺陷）的成纤维细胞培养物匀浆，未检测到对[氨基磺酰基-³⁵S]肝素、单糖、二糖或四糖底物的可检测氨磺酰酶活性。(5) 用GlcNS-IdOA测量的氨磺酰酶活性在pH 4.5至5.5时表现出最佳值，表观Km约为220μmol/L，并受到硫酸根离子的强烈抑制。
