Freeman C, Clements P R, Hopwood J J
Department of Chemical Pathology, Adelaide Children's Hospital, Australia.
Biochem J. 1987 Sep 1;246(2):347-54. doi: 10.1042/bj2460347.
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.
人N-乙酰葡糖胺-6-硫酸酯硫酸酯酶通过一种简单的三步四柱程序从肝脏中纯化至至少50000倍的纯度且产率为78%，达到了均一性。该程序包括伴刀豆球蛋白A-琼脂糖/蓝色A-琼脂糖偶联步骤、色谱聚焦和Cu²⁺-螯合琼脂糖色谱法。总共分离出四种形式并对其进行了部分表征。A和B两种形式，其pI均大于9.5，分别占回收酶活性的30%和60%，通过对从Cu²⁺-螯合琼脂糖步骤获得的酶制剂进行羟基磷灰石色谱法分离。A和B两种形式的天然分子量均为75 kDa。通过SDS/聚丙烯酰胺凝胶电泳分析时，A形式由一条分子量为78 kDa的单一多肽组成，而B形式包含48 kDa和32 kDa的多肽亚基。A和B两种形式均未从培养基被摄取到培养的人皮肤成纤维细胞中。另外两种形式（C和D），pI值分别为5.8和5.4，分别占总回收酶活性的约7%和3%。C和D两种形式的天然分子量分别为94 kDa和约75 kDa。C形式包含三条分子量分别为48、45和32 kDa的多肽。N-乙酰葡糖胺-6-硫酸酯硫酸酯酶活性用一种源自肝素的放射性标记二糖底物进行测定。这种底物的开发使得N-乙酰葡糖胺-6-硫酸酯硫酸酯酶的分离和表征能够高效进行。A、B和C三种形式的最适pH为5.0，Km值分别为11.7、14.2和11.1 μM，Vmax值分别为每毫克蛋白质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，这四种酶形式在结构和性质上的差异是由于一个大亚基加工状态的差异所致。
