Maksimenko A V, Schechilina Y V, Tischenko E G
Institute of Experimental Cardiology, Russian Cardiology Research Center, Moscow 121552, Russia.
Biochemistry (Mosc). 2003 Aug;68(8):862-8. doi: 10.1023/a:1025794830705.
The glycosaminoglycan microenvironment of testicular hyaluronidase was simulated by multipoint covalent attachment of the enzyme to glycans as a result of benzoquinone activation. The efficiency of their binding was assessed using gel chromatography, ultrafiltration, titration of surface amino groups of the enzyme, electrophoresis, as well as judging by the value of residual endoglycosidase activity and its inhibition with heparin. Copolymer glycosaminoglycans, such as dermatan sulfate and heparin, inactivated the endoglycosidase activity as a result the C-5 epimerization of hexuronic acid. It was shown that glucuronic acid and, to a lesser extent, N-acetylglucosamine determine the specificity of hyaluronidase. The chondroitin-sulfate microenvironment made the enzyme resistant to heparin inhibition because the equatorial orientation of the OH groups is similar to that in hyaluronic acid. Model experiments with dextran and dextran sulfate showed that sulfation of the glycan chain increased its rigidity, thus hampering the stabilizing effect on hyaluronidase. The effect of chondroitin sulfate on the endoglycosidase activity of hyaluronidase had additive character and did not directly affect the small fragment of the active site of the enzyme located at the bottom of a groove. The glycosaminoglycan microenvironment of hyaluronidase, containing an iduronic acid residue, the alpha1-3 and alpha1-4 glycosidic bond, inactivated the hyaluronidase activity of the enzyme, whereas simple polymers (such as gluco- and galactoaminoglycans) potentiated it due to a similar way of linking--beta(1e-4e) and beta(1e-3e). To understand the nature of these interactions in detail, the effect of oligomeric glycosaminoglycan fragments and their derivatives on hyaluronidase should be studied.
通过苯醌活化使睾丸透明质酸酶与聚糖多点共价连接，模拟了睾丸透明质酸酶的糖胺聚糖微环境。使用凝胶色谱、超滤、酶表面氨基滴定、电泳以及通过残留内切糖苷酶活性值及其对肝素的抑制作用来评估它们的结合效率。硫酸皮肤素和肝素等共聚物糖胺聚糖由于己糖醛酸的C-5差向异构化而使内切糖苷酶活性失活。结果表明，葡萄糖醛酸以及程度稍低的N-乙酰葡糖胺决定了透明质酸酶的特异性。硫酸软骨素微环境使该酶对肝素抑制具有抗性，因为羟基的赤道取向与透明质酸中的相似。用葡聚糖和硫酸葡聚糖进行的模型实验表明，聚糖链的硫酸化增加了其刚性，从而妨碍了对透明质酸酶的稳定作用。硫酸软骨素对透明质酸酶内切糖苷酶活性的影响具有加和性，并且不直接影响位于凹槽底部的酶活性位点的小片段。含有艾杜糖醛酸残基、α1-3和α1-4糖苷键的透明质酸酶的糖胺聚糖微环境使该酶的透明质酸酶活性失活，而简单聚合物（如葡糖胺聚糖和半乳糖胺聚糖）由于类似的连接方式——β(1e-4e)和β(1e-3e)而增强了它的活性。为了详细了解这些相互作用的本质，应该研究寡聚糖胺聚糖片段及其衍生物对透明质酸酶的影响。
