Xanthine oxidase binding to glycosaminoglycans: Kinetics and superoxide dismutase interactions of immobilized xanthine oxidase-heparin complexes
Xanthine oxidase binding to glycosaminoglycans: Kinetics and superoxide dismutase interactions of immobilized xanthine oxidase-heparin complexes
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DOI：
10.1006/abbi.1996.9844
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发表时间：
1997-03-01
影响因子：
3.9
通讯作者：
Freeman, BA
中科院分区：
文献类型：
--
作者：
Radi, R;Rubbo, H;Freeman, BA
黄嘌呤氧化还原酶（XDH + XO，EC 1.2.3.2）从富含XO活性的器官释放到循环中，在此我们报道了XO与糖胺聚糖（GAG）的特异性高亲和力结合以及XO与肝素的优先结合，与硫酸乙酰肝素、硫酸软骨素和硫酸地马坦相比。XO与Sepharose 6 B结合肝素（HS 6 B）的结合发生在生理离子强度下，并随pH值增加而增加，Scatchard分析显示在pH 7.4时为非线性结合模式。XO结合的解离常数（Kd）为0.4至1.8 x 10（-7）M，与脂蛋白脂肪酶与血管内皮的肝素可逆结合相似。结合能为9-13 kcal/mol，与非共价静电相互作用一致。黄嘌呤氧化酶固定到HS 6 B上，得到了具有催化活性的酶形式，其动力学特性与游离溶液中的XO不同。在pH7.4的磷酸盐缓冲液中，黄嘌呤的Km和Ki分别为3 μ M和1.6mM，而游离XO的Km和Ki分别为15 μ M和2.8mM，鸟嘌呤和尿酸的抑制常数也增加XO结合HS 6 B。动力学参数的变化与底物和抑制剂的结合亲和力的真实的而非明显的降低有关，而不是由于凝胶基质内的扩散控制过程。黄嘌呤的K-m和K-i的变化也对O-2（.-）的相对量有显著影响。以及由给定底物浓度产生的H2 O2。HS 6 B结合XO形成的超氧化物部分消耗的凝胶内的微环境，静电排除铜锌SOD。XO的固定化增加了酶活性在缓冲液中的半衰期，并在没有底物的情况下，在4 ℃下从67小时至120小时。这些数据表明，结合到细胞表面将强烈影响XO的催化性能，氧化剂产生能力和稳定性。(C)北京：科学出版社.
Xanthine oxidoreductase (XDH + XO, EC 1.2.3.2) is released into the circulation from organs rich in XO activity, Herein we report the specific high affinity binding of XO to glycosaminoglycans (GAGs) and the preferential association of XO with heparin, compared with heparan sulfate, chondroitin sulfate, and dematan sulfate. The binding of XO to Sepharose 6B-conjugated heparin (HS6B) occurs at physiological ionic strength and increased with pH, with Scatchard analysis revealing a nonlinear binding pattern at pH 7.4, The dissociation constant (K-d) for XO binding was 0.4 to 1.8 x 10(-7) M, similar to the heparin-reversible binding of lipoprotein lipase to vascular endothelium. The binding energy of 9-13 kcal/mol was concordant with noncovalent electrostatic interactions. Xanthine oxidase immobilization to HS6B rendered a catalytically active enzyme form that had kinetic characteristics distinct from XO in free solution, While the K-m and K-i for xanthine in phosphate buffer at pH 7.4 were 3 mu M and 1.6 mM, respectively, for free XO, they were 15 mu M and 2.8 mM for immobilized XO, Inhibition constants for guanine and uric acid were also increased upon XO binding to HS6B. Changes in kinetic parameters were related to a real and not apparent decrease in binding affinity for substrate and inhibitors and were not due to diffusion-controlled processes within the gel matrix, Changes in K-m and K-i for xanthine also had a significant influence on the relative quantities of O-2(.-) and H2O2 generated by a given substrate concentration. Superoxide formed by HS6B-bound XO was partially consumed within the gel microenvironment which electrostatically excluded CuZn SOD. Immobilization of XO increased the half life of enzyme activity in buffer and in the absence of substrate from 67 to 120 h at 4 degrees C, These data indicate that binding to cell surfaces will strongly influence the catalytic properties, oxidant producing capacity, and stability of XO. (C) 1997 Academic Press.