Gu K, Linhardt R J, Laliberté M, Gu K, Zimmermann J
Division of Medicinal and Natural Products Chemistry, College of Pharmacy, University of Iowa, Iowa City 52242, USA.
Biochem J. 1995 Dec 1;312 ( Pt 2)(Pt 2):569-77. doi: 10.1042/bj3120569.
The chondroitin lyases from Flavobacterium heparinum (Cytophaga heparinia) have been widely used in depolymerization of glycosaminoglycan and proteoglycan chondroitin sulphates. Oligosaccharide products derived from chondroitin sulphate can be further degraded by glycuronidases and sulphatases obtained from the same organism. There has been no reported purification of these enzymes to homogeneity nor is there any information on their physical and kinetic characteristics. The absence of pure enzymes has resulted in a lack of understanding of the optimal conditions for their catalytic activity and their substrate specificity. This has limited the use of these enzymes as reagents for preparation of oligosaccharides for structure and activity studies. Reproducible schemes to purify a chondroitin AC lyase, a glycuronidase and chondroitin B lyase from Flavobacterium heparinum to apparent homogeneity are described. Chondroitin AC lyase (chondroitinase AC, EC 4.2.2.5), glycuronidase [chondro-(1-->3)-glycuronidase, no EC number] and chondroitin B lyase (chondroitinase B, no EC number) have M(r) values (assessed by SDS/PAGE) of 74,000, 41,800 and 55,200 respectively, and isoelectric points (determined by isoelectric focusing) of 8.85, 9.28 and 9.05 respectively. Chondroitin lyase AC and B contain pyroglutamic acid at their N-termini precluding their analysis by Edman degradation. Deblocking with pyroglutamate aminopeptidase facilitated the determination of their N-terminal sequences. The kinetic properties of these enzymes have been determined as well as the optimum conditions for their catalytic activity. The specificity of the glycouronidase, determined using 17 different disaccharide substrates, shows that it only acts on unsulphated or 6-O-sulphated 1-->3 linkages. The chondroitin lyases are both endolytic enzymes, and oligosaccharide mapping shows their expected specificity towards the chondroitin and dermatan sulphate polymers.
来自肝素黄杆菌（食蟹黄杆菌）的软骨素裂解酶已被广泛用于糖胺聚糖和蛋白聚糖硫酸软骨素的解聚。硫酸软骨素衍生的寡糖产物可被从同一生物体获得的葡萄糖醛酸酶和硫酸酯酶进一步降解。目前尚无关于这些酶纯化至均一性的报道，也没有关于它们的物理和动力学特性的任何信息。缺乏纯酶导致对其催化活性的最佳条件及其底物特异性缺乏了解。这限制了这些酶作为制备用于结构和活性研究的寡糖的试剂的应用。本文描述了从肝素黄杆菌中纯化软骨素AC裂解酶、葡萄糖醛酸酶和软骨素B裂解酶至明显均一性的可重复方案。软骨素AC裂解酶（软骨素酶AC，EC 4.2.2.5）、葡萄糖醛酸酶[软骨素-(1→3)-葡萄糖醛酸酶，无EC编号]和软骨素B裂解酶（软骨素酶B，无EC编号）的相对分子质量（通过SDS/PAGE评估）分别为74,000、41,800和55,200，等电点（通过等电聚焦测定）分别为8.85、9.28和9.05。软骨素裂解酶AC和B在其N端含有焦谷氨酸，这使得无法通过埃德曼降解法对其进行分析。用焦谷氨酸氨肽酶去封闭有助于确定它们的N端序列。已测定了这些酶的动力学性质以及它们催化活性的最佳条件。使用17种不同的二糖底物确定的葡萄糖醛酸酶的特异性表明，它仅作用于未硫酸化或6-O-硫酸化的1→3连接。软骨素裂解酶都是内切酶，寡糖图谱显示了它们对软骨素和硫酸皮肤素聚合物的预期特异性。
