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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 ,且ADAbp对ADA活性有抑制作用
Objective: To investigate the molecular characteristics of human renal cell adenosine deaminase (ADA). Methods: ADA was isolated and purified by immunoaffinity chromatography, and the activity of ADA was determined by ammonia assay. Results: The obtained ADA holoenzyme reached electrophoretic purity with an enzyme specific activity of 25 6 1.41 U / mg and a yield of 26.59%. By SDS PAGE analysis, the holoenzyme consists of a 45.6 KD small molecule ADA subunit and an ADA-free binding protein (ADAbp, with molecular weights of 6 8.1 KD and 10 8.7 KD). With adenosine or deoxyadenosine as substrates, the holoenzyme Km values were 89μmol / L and 67μmol / L, respectively. P-mercapto benzoic acid can significantly inhibit enzyme activity, dithiothreitol can be inhibited activity was partially restored, when the concentration of adenosine in the range of 50 ~ 150μmol / L, ADAbp inhibition of ADA activity was 18.4% ~ 2 1.5%. CONCLUSION: ADA is a macromolecular ADA composed of small molecule ADA subunit and ADAbp without ADA in human renal cell. Sulfhydryl group is an essential group of the active center, and ADAbp can inhibit ADA activity