Paschke E, Kresse H
Biochem J. 1979 Sep 1;181(3):677-84. doi: 10.1042/bj1810677.
2-Deoxyglucoside-2-sulphamate sulphohydrolase was purified about 10 000-fold from the soluble extract of human placenta by using as substrate [N-sulpho-35S]heparin. Differently charged enzyme forms were observed on chromatography on DEAE-cellulose, all of which had an apparent mol.wt. of 110 000 as determined by gel filtration. By using immobilized heparan sulphate as affinity matrix the sulphamate sulphohydrolase could be separated into two forms, a minor one with low and a major one with high affinity for the adsorbent. When tested with [N-sulpho-35S]heparan sulphate the low-affinity form had a Km of 0.2 mM, and the high-affinity form a Km of 0.03 mM. Both forms exhibited the same Km of 10 microM towards [N-sulpho-35S]heparin and were equally well adsorbed to immobilized heparin. The two forms could be distinguished by their pH-optima and by the influence of KCl on heparan sulphate sulphohydrolase activity.
以[N-磺基-35S]肝素为底物，从人胎盘的可溶性提取物中纯化出2-脱氧葡萄糖苷-2-磺酸酯硫酸酯酶，纯化倍数约为10000倍。在DEAE-纤维素柱层析上观察到电荷不同的酶形式，通过凝胶过滤测定，所有这些酶形式的表观分子量均为110000。以固定化硫酸乙酰肝素为亲和基质，可将磺酸酯硫酸酯酶分离为两种形式，一种对吸附剂亲和力低的次要形式和一种对吸附剂亲和力高的主要形式。用[N-磺基-35S]硫酸乙酰肝素进行测试时，低亲和力形式的Km为0.2 mM，高亲和力形式的Km为0.03 mM。两种形式对[N-磺基-35S]肝素的Km均为10 μM，并且对固定化肝素的吸附效果相同。这两种形式可通过其最适pH值以及KCl对硫酸乙酰肝素硫酸酯酶活性的影响来区分。
