肾腺苷脱氨酶及其结合蛋白的纯化和性质研究

Purification and Characterization of Adenosine Deaminase and Adenosine Deaminase Binding Protein from Human Kidney

目的 :探讨人肾细胞腺苷脱氨酶 (ADA)分子特性。方法 :用免疫亲和层析法分离纯化ADA ,测氨法测定ADA活性。结果 :获得的ADA全酶分子达电泳纯 ,酶比活力为 2 5 6 1.41U/mg ,得率 2 6 .5 9%。经SDS PAGE分析 ,全酶由 45 .6KD的小分子的ADA亚单位和无ADA活性的结合蛋白 (ADAbp ,其分子量为 6 8.1KD和 10 8.7KD)构成。用腺苷或脱氧腺苷为底物 ,全酶的Km值分别为 89μmol/L和 6 7μmol/L。对氯汞苯甲酸能显著抑制酶活性 ,二硫苏糖醇可使被抑制的活性得到部分恢复 ,当腺苷浓度在 5 0～ 15 0 μmol/L范围内 ,ADAbp对ADA活性的抑制率为 18.4%～ 2 1.5 %。结论 :人肾细胞中ADA为小分子ADA亚单位和无ADA活性的ADAbp构成的大分子ADA ,巯基是该酶活性中心的必需基团 。

Objective: To study molecular properties of the adenosine deaminase (ADA) in cells of human kidney. Methods: Adenosine deaminase was isolated and purified using immuno_affinity chromatography,the enzyme activity was assayed colorimetrically by the measurement of ammonia produced over a fixed time. Results: The purified ADA was shown to be a homogeneity by polyacrylamine gel electrophoresis (PAGE).The yield and specific activity of purified ADA were 26.59% and 2 561.4 (U/mg) respetively.the purified ADA molecule consists of 45.6 KD Mr small subunit ADA、inactive 68.1 KD Mr and 108.7 KD Mr binding protein (ADAbp) on a 10% SDS_PAGE.Using adenosine or 2_deoxyadenosine as substrate the apparent Km of the enzyme is 89 μmol/L and 67 μmol/L respectively.The enzyme activity can be inhibited by p_chloromecuribenzoate while partially restored by dithiothreitol.The isolated ADAbp can inhibit small subunit ADA activity by 18.4%～21.5% during the adenosine concentration of 50～150 μmol/L. Conclusion: Adenosine deaminase from human kidney is a complex consisting of small subunit ADA and inactive ADA bp.The hydrosulfuryl is essential group in the active center of the enzyme,and ADA bp can inhibit the catalytic activity of small subunit ADA.