Sugahara K, Nadanaka S, Takeda K, Kojima T
Department of Biochemistry, Kobe Pharmaceutical University, Japan.
Eur J Biochem. 1996 Aug 1;239(3):871-80. doi: 10.1111/j.1432-1033.1996.0871u.x.
The enzymatic action of highly purified chondroitin ABC lyase from Proteus vulgaris is dependent on the size of the substrate, and the enzyme does not cleave tetrasaccharides, irrespective of their sulfation profiles [Sugahara, K., Shigeno, K., Masuda, M., Fujii, N., Kurosaka, A. & Takeda, K. (1994) Carbohydr. Res. 255, 145-163]. To characterize the enzyme action in more detail, we isolated nine sulfated hexasaccharides from commercial shark cartilage chondroitin sulfate D, after partial digestion with highly purified chondroitin ABC lyase, by means of gel chromatography and HPLC on an amine-bound silica column. Structural analysis by 500-MHz H-NMR spectroscopy, and enzymatic digestion in conjunction with HPLC, demonstrated that these hexasaccharides, with the common core saccharide structure delta 4 HexA (alpha 1-3)GalNAc(beta 1-4)GlcA(beta 1-3)GalNAc(beta 1-4) GlcA(beta 1-3)GalNAc(where delta 4 HexA and GlcA represent 4-deoxy-alpha-L-threo-hex-4-enepyranosyluronic acid and glucuronic acid, respectively) bear three or four sulfate groups in different combinations. In the hexasaccharides, the D, disaccharide unit GlcA2-SO3 (beta 1-3) GalNAc4SO(3-) which is characteristic of chondroitin sulfate D, was arranged on the reducing side of the A disaccharide unit GlcA(beta 1-3)GalNAc4SO(3)-, and thus formed an A-D tetrasaccharide sequence GlcA(beta 1-3)GalNAc4SO(3)-(beta 1-4)GlcA2SO(3)-(beta 1-3) GalNAc6SO(3)-. Analysis of the degradation products of these hexasaccharides with highly purified chondroitin ABC lyase indicated that the enzyme preferentially acted on the unsaturated hexasaccharides in an exolytic fashion and removed an unsaturated disaccharide unit from the non-reducing termini, irrespective of the sulfation profiles of the hexasaccharides.
普通变形杆菌中高度纯化的软骨素ABC裂解酶的酶促作用取决于底物的大小，并且该酶不会切割四糖，无论其硫酸化模式如何[Sugahara, K., Shigeno, K., Masuda, M., Fujii, N., Kurosaka, A. & Takeda, K. (1994) Carbohydr. Res. 255, 145 - 163]。为了更详细地描述该酶的作用，我们在用高度纯化的软骨素ABC裂解酶进行部分消化后，通过凝胶色谱法和胺基键合硅胶柱上的高效液相色谱法，从商业鲨鱼软骨硫酸软骨素D中分离出九种硫酸化六糖。通过500兆赫的氢核磁共振光谱进行结构分析，并结合高效液相色谱法进行酶促消化，结果表明这些六糖具有共同的核心糖结构δ4 HexA（α1 - 3）GalNAc（β1 - 4）GlcA（β1 - 3）GalNAc（β1 - 4）GlcA（β1 - 3）GalNAc（其中δ4 HexA和GlcA分别代表4 - 脱氧-α-L-苏糖-己-4-烯吡喃糖醛酸和葡萄糖醛酸），带有三种或四种不同组合的硫酸基团。在这些六糖中，硫酸软骨素D特有的二糖单元GlcA2 - SO3（β1 - 3）GalNAc4SO(3-)排列在二糖单元GlcA（β1 - 3）GalNAc4SO(3)-的还原端，从而形成了A - D四糖序列GlcA（β1 - 3）GalNAc4SO(3)-（β1 - 4）GlcA2SO(3)-（β1 - 3）GalNAc6SO(3)-。用高度纯化的软骨素ABC裂解酶对这些六糖的降解产物进行分析表明，该酶优先以外切方式作用于不饱和六糖，并从非还原末端去除一个不饱和二糖单元，而不考虑六糖的硫酸化模式。
