Bruce J S, McLean M W, Long W F, Williamson F B
Eur J Biochem. 1985 Apr 15;148(2):359-65. doi: 10.1111/j.1432-1033.1985.tb08847.x.
A novel bacterial sulphatase has been discovered in an extract of Flavobacterium heparinum. The enzyme hydrolyses the 3-O-sulphate from 2-deoxy-2-sulphamido-3-O-sulpho-D-glucose and 2-acetamido-2-deoxy-3-O-sulpho-D-glucose. The activity was purified 10 800-fold by chromatography successively on CM-Sepharose CL-6B, hydroxyapatite, taurine-Sepharose CL-4B and CM-Sepharose CL-6B. Sodium dodecylsulphate/polyacrylamide gel electrophoresis showed the enzyme to be homogeneous and of relative molecular mass 56 000. Two novel assays were developed using 2-[14C]acetamido-2-deoxy-3-O-sulpho-D-glucose and 2-deoxy-2-sulphamido-3-O-sulpho-D-glucose as respective substrates. The purified 3-O-sulphatase was shown to be free of all other known heparin-degrading enzymes. Optimal activity was at pH 7.5 for the disulphated substrate and pH 8.0 for the N-acetylated substrate. Enzyme activity was virtually unaffected by Na+, K+ or Mg2+ ions. A 1.2-fold enhancement of activity was effected by 0.002 mol dm-3 Ca2+. Inorganic phosphate and sulphate inhibited 3-O-sulphatase activity. The Km value of the N-acetylated substrate was determined to be 42 mumol dm-3. No activity was detected with 2-amino-2-deoxy-3-O-sulpho-D-glucose.
在肝素黄杆菌提取物中发现了一种新型细菌硫酸酯酶。该酶可从2-脱氧-2-氨磺酰基-3-O-磺基-D-葡萄糖和2-乙酰氨基-2-脱氧-3-O-磺基-D-葡萄糖中水解3-O-硫酸盐。通过依次在CM-琼脂糖凝胶CL-6B、羟基磷灰石、牛磺酸-琼脂糖凝胶CL-4B和CM-琼脂糖凝胶CL-6B上进行色谱分离，该活性被纯化了10800倍。十二烷基硫酸钠/聚丙烯酰胺凝胶电泳显示该酶是均一的，相对分子质量为56000。使用2-[14C]乙酰氨基-2-脱氧-3-O-磺基-D-葡萄糖和2-脱氧-2-氨磺酰基-3-O-磺基-D-葡萄糖分别作为底物，开发了两种新的测定方法。纯化的3-O-硫酸酯酶被证明不含所有其他已知的肝素降解酶。对于双硫酸化底物，最佳活性在pH 7.5，对于N-乙酰化底物，最佳活性在pH 8.0。酶活性几乎不受Na+、K+或Mg2+离子的影响。0.002 mol dm-3的Ca2+使活性提高了1.2倍。无机磷酸盐和硫酸盐抑制3-O-硫酸酯酶活性。N-乙酰化底物的Km值测定为42 μmol dm-3。用2-氨基-2-脱氧-3-O-磺基-D-葡萄糖未检测到活性。
