人胃腺苷脱氨酶的分离纯化及性质研究

用硫酸铵分级沉淀、ＤＥＡＥ－纤维素离子交换层析、免疫亲和层析、ＳｅｐｈａｄｅｘＧ１００凝胶柱层析从人胃组织中提取出腺苷脱氨酶，酶纯化１９３２４倍，比活力为５７９７Ｕ／ｍｇ蛋白．提取酶液经ＰＡＧＥ、ＳＤＳ－ＰＡＧＥ和等电聚焦只呈现一条区带。测得该酶的分子量为４１．２ｋＤ，等电点为ｐＨ４．８．氨基酸组成分析表明该酶由３８８个氨基酸残基组成，Ｎ端氨基酸为精氨酸。酶的最适ｐＨ为６．５，ｐＨ小于５．０或大于９．０时不稳定；最适温度为３７℃，对热不太稳定，以腺苷及２－脱氧腺苷作为底物，其Ｋｍ分别为８７μｍｏｌ／Ｌ和４１μｍｏｌ／Ｌ。

Studies on Purification and Properties of Adenosine Deaminase from Human Stomach

Adenosine deaminase(EC 3.5.4.4.,ADA) was purified from human stomach bx the procedures including ammonium sulfate differential precipitation,DEAE-cellulose chromatography immuno-affinity chromatography and Sephadex G-100 gel filtration.Compared with the crude extract,the resulting enzyme solution was purified by 19324 fold,and its specific activity was 5797 units/mg protein.The purified ADA showed single protein band on gels of PAGE,SDS-PAGE and isoelectric focusing separately,which indicated a molecular weight of 41.2 kD on SDS-PAGE gel and at pH4.8 on electric focusing gel.The resulting enzyme consists of about 388 amino acid residues with arginine as Nteminal.The Km for adenosine and Km for deoxyadenosine are 87μmol/L and 41μmol/L,respectively.The optimum temperature is 37℃ and the optimum pH is 6.5.