EXAMINATION OF THE SUBSTRATE-SPECIFICITY OF HEPARIN AND HEPARAN-SULFATE LYASES
EXAMINATION OF THE SUBSTRATE-SPECIFICITY OF HEPARIN AND HEPARAN-SULFATE LYASES
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DOI：
10.1021/bi00462a026
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发表时间：
1990-03-13
期刊：
影响因子：
2.9
通讯作者：
GALLAGHER, JT
中科院分区：
文献类型：
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作者：
LINHARDT, RJ;TURNBULL, JE;GALLAGHER, JT
我们考察了不同制剂的肝素和硫酸肝素裂解酶的活性。这些酶与已知大小和顺序的寡糖以及复杂的多糖底物孵育，得到的降解产物用强阴离子交换高效液相色谱和低聚糖梯度凝胶电泳法进行分析。我们实验室纯化的肝素酶(EC 4.2.2.7)和商业来源的所谓的肝素酶I(Hep I)产生了与肝素底物相似的寡糖图谱，并显示出对结构为.fwdarw的二硫或三硫双糖的特异性。4)-.alpha.-D-GlcNp2S(6R)(1.fwdarw.-4)-.alpha.-L-IdoAp2S(1.fwdarw.(式中R=0-磺酸或羟基)。两种不同商品化的硫酸肝素裂解酶[肝素酶(EC 4.2.2.8)]的寡糖图谱显示，它们对硫酸乙酰肝素的解聚非常相似。此外，这些酶只降解与葡萄糖醛酸连接的已糖胺键上的特定寡糖：.fwdarw.4)-.alpha.-D-GlcNpR(1.fwdarw.4)-.beta.-D-GlcAp(1.fwdarw.(式中R=N-乙酰胺或N-磺酸)。这些酶在其他高度硫酸盐化的区域显示出对单独的含葡萄糖醛酸二糖的活性，包括含有肝素中抗凝血酶结合区的糖序列。另一种不同的商业酶，肝素酶II(Hep II)，对多糖和低聚糖底物显示出广泛的活性，但图谱数据表明它是一个单独的酶，而不是肝素酶和肝素酶/Hep III的混合物。当与所描述的分离程序结合使用时，这些酶是分析肝素和硫酸肝素的结构/序列的强大试剂。
We have examined the activities of different preparations of heparin and heparan sulfate lysases from Flavobacterium heparinum. The enzymes were incubated with oligosaccharides of known size and sequence and with complex polysaccharide substrates, and the resulting degradation products were analyzed by strong-anion-exchange high-performance liquid chromatography and by oligosaccharide mapping using gradient polyacrylamide gel electrophoresis. Heparinase (EC 4.2.2.7) purified in our laboratory and so-called Heparinase I (Hep I) from a commercial source yielded similar oligosaccharide maps with heparin substrates and displayed specificity for di- or trisulfated disaccharides of the structure .fwdarw. 4)-.alpha.-D-GlcNp2S(6R)(1.fwdarw.-4)-.alpha.-L-IdoAp2S(1.fwdarw. (where R = 0-sulfo or OH). Oligosaccharide mapping with two different commercial preparations of heparan sulfate lysase [heparitinase (EC 4.2.2.8)] indicated close similarities in their depolymerization of heparan sulfate. Furthermore, these enzymes only degraded defined oligosaccharides at hexosaminidic linkages with glucuronic acid: .fwdarw.4)-.alpha.-D-GlcNpR(1.fwdarw.4)-.beta.-D-GlcAp(1.fwdarw. (where R = N-acetamido or N-sulfo). The enzymes showed activity against solitary glucuronate-containing disaccharides in otherwise highly sulfated domains including the saccharide sequence that contains the antithrombin binding region in heparin. A different commercial enzyme, Heparinase II (Hep II), displayed a broad spectrum of activity against polysaccharide and oligosaccharide substrates, but mapping data indicated that it was a separate enzyme rather than a mixture of heparinase and heparitinase/Hep III. When used in conjunction with the described separation procedures, these enzymes are powerful reagents for the structural/sequence analysis of heparin and heparan sulfate.