Human liver N-acetylglucosamine-6-sulphate sulphatase. Purification and characterization.
Human liver N-acetylglucosamine-6-sulphate sulphatase. Purification and characterization.
复制标题
人肝 N-乙酰氨基葡萄糖-6-硫酸硫酸酯酶。
DOI：
10.1042/bj2460347
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发表时间：
1987
期刊：
影响因子：
--
通讯作者：
J. Hopwood
中科院分区：
文献类型：
--
作者：
C. Freeman;P. Clements;J. Hopwood
通过简单的三步四柱程序（包括伴刀豆球蛋白 A-琼脂糖/蓝 A-琼脂糖偶联步骤、色谱聚焦和 Cu2+ 螯合琼脂糖层析），从肝脏中将人 N-乙酰氨基葡萄糖-6-硫酸酯硫酸酯酶纯化至少 50,000 倍，达到同质性，收率 78%。总共有四种形式被分离出来并得到部分表征。 A 型和 B 型的 pI 都大于 9.5，分别代表恢复的酶活性的 30% 和 60%，通过从 Cu2+ 螯合琼脂糖步骤获得的酶制剂的羟基磷灰石色谱分离。 A 型和 B 型的天然分子质量均为 75 kDa。当通过SDS/聚丙烯酰胺凝胶电泳分析时，形式A由分子量78kDa的单一多肽组成，而形式B包含48kDa和32kDa多肽亚基。 A型和B型均未从培养基中吸收到培养的人皮肤成纤维细胞中。其他两种形式（C 和 D）的 pI 值分别为 5.8 和 5.4，代表大约 10%。总恢复酶活性的7%和3%。 C型和D型的天然分子质量为94 kDa，约为10 kDa。分别为 75 kDa。 C型含有三种多肽，分子量分别为48、45和32 kDa。 N-乙酰葡糖胺-6-硫酸盐硫酸酯酶活性使用源自肝素的放射性标记二糖底物进行测量。该底物的开发使得 N-乙酰氨基葡萄糖-6-硫酸酯硫酸酯酶的分离和表征能够有效地进行。形式A、B和C的最适pH为5.0，Km值分别为11.7、14.2和11.1μM，Vmax。每毫克蛋白质的值分别为 105、60 和 53 nmol/min。这种硫酸酯酶多种形式的分子基础尚不清楚。据推测，四种酶形式的结构和性质的差异是由于大亚基的加工状态的差异造成的。
Human N-acetylglucosamine-6-sulphate sulphatase was purified at least 50,000-fold to homogeneity in 78% yield from liver with a simple three-step four-column procedure, which consists of a concanavalin A-Sepharose/Blue A-agarose coupled step, chromatofocusing and Cu2+-chelating Sepharose chromatography. In all, four forms were isolated and partially characterized. Forms A and B, both with a pI greater than 9.5 and representing 30% and 60% respectively of the recovered enzyme activity, were separated by hydroxyapatite chromatography of the enzyme preparation obtained from the Cu2+-chelating Sepharose step. Both forms A and B had native molecular masses of 75 kDa. When analysed by SDS/polyacrylamide-gel electrophoresis, form A consists of a single polypeptide of molecular mass 78 kDa, whereas form B contained 48 kDa and 32 kDa polypeptide subunits. Neither form A nor form B was taken up from the culture medium into cultured human skin fibroblasts. The two other forms (C and D), with pI values of 5.8 and 5.4 respectively, represented approx. 7% and 3% of the total recovered enzyme activity. The native molecular masses of forms C and D were 94 kDa and approx. 75 kDa respectively. Form C contained three polypeptides with molecular masses of 48, 45 and 32 kDa. N-Acetylglucosamine-6-sulphate sulphatase activity was measured with a radiolabelled disaccharide substrate derived from heparin. The development of this substrate enabled the isolation and characterization of N-acetylglucosamine-6-sulphate sulphatase to proceed efficiently. Forms A, B and C had pH optima of 5.0, Km values of 11.7, 14.2 and 11.1 microM respectively and Vmax. values of 105, 60 and 53 nmol/min per mg of protein respectively. The molecular basis of the multiple forms of this sulphatase is not known. It is postulated that the differences in structure and properties of the four enzyme forms are due to differences in the state of processing of a large subunit.