一种S-腺苷甲硫氨酸依赖的甲基转移酶活性的检测方法

摘要

通过PCR扩增获得了S-腺苷-L-高半胱氨酸核苷酶(SAHN)和S-核糖基高半胱氨酸酶(SRHH)的基因序列,克隆入表达载体,转化宿主细胞,表达纯化得到带有组氨酸标签的重组蛋白,基于SAHN和SRHH重组酶建立了一种酶偶联分析S-腺苷甲硫氨酸(AdoMet)依赖的甲基转移酶活性的检测方法.甲基转移酶的共同产物S-腺苷-L-高半胱氨酸(AdoHcy)首先被SAHN酶水解生成腺嘌呤和S-核苷高半胱氨酸,后者进一步被SRHH酶裂解生成高半胱氨酸,最后高半胱氨酸与Ellman's试剂反应生成5-硫代-2-硝基苯甲酸(TNB).实验结果表明,重组表达获取的2种酶蛋白均具有良好的催化活性,酶偶联反应生成的TNB与初始AdoHcy浓度呈现显著的线性正相关.该检测方法克服了S-腺苷甲硫氨酸依赖的甲基转移酶的共同产物AdoHcy的反馈抑制,使甲基化反应进行完全,从而保证了对甲基转移酶活性准确有效的定量分析.%S-Adenosyl-L-methionine ( AdoMet) -dependent methyltransferases are a widespread class of enzymes exist in both prokaryotes and eukaryotes. Here we report a coupled enzymatic analysis method established to quantitatively characterize S-adenosyl-L-homocysteine ( AdoHcy) , the common product of AdoMet-de-pendent methyltransferases. Recombinant S-adenosylhomocysteine nucleosidase ( SAHN) and recombinant S-ribosylhomocysteinase(SRHH) , which are involved in the enzymatic method, were cloned and expressed as His-tag proteins, and further purified with Ni2+ charged agarose resin column, respectively. In this assay, AdoHcy was first hydrolyzed by SAHN into adenine and S-ribosylhomocysteine. Subsequently, the compound S-ribosylhomocysteine generated from SAHN was further cleaved by SRHH to form homocysteine. Finally, ho-mocysteine was quantified using DTNB ( Ellman' s reagent) . The experimental result demonstrated a fine linear positive correlation between formed TNB and initial concentration of AdoHcy, and repeatability was very good. The advantage of this methyltransferase analysis to traditional radioactivity assay is the removal of AdoHcy product by SAHN, which alleviates product feedback inhibition of AdoMet-dependent methyltransferases. This method may be used to analyze other enzymes that produce AdoHcy.