一种用于 S-腺苷甲硫氨酸依赖性甲基转移酶的酶联比色测定法。
An enzyme-coupled colorimetric assay for S-adenosylmethionine-dependent methyltransferases.
作者信息
Hendricks Cheryl L, Ross Jeannine R, Pichersky Eran, Noel Joseph P, Zhou Zhaohui Sunny
机构信息
Department of Chemistry, Carnegie Mellon University, Pittsburgh, PA 15213, USA.
出版信息
Anal Biochem. 2004 Mar 1;326(1):100-5. doi: 10.1016/j.ab.2003.11.014.
We report here an enzyme-coupled colorimetric assay for salicylic acid carboxyl methyltransferase (SAMT), which utilizes S-adenosyl-l-methionine (AdoMet or SAM) as the methyl donor. In this assay, S-adenosyl-l-homocysteine (AdoHcy or SAH), a common product of AdoMet-dependent transmethylation reactions, is first hydrolyzed by recombinant AdoHcy nucleosidase (EC 3.2.2.9) into adenine and S-ribosylhomocysteine. Recombinant LuxS (S-ribosylhomocysteinase, EC 3.2.1.148) cleaves the latter compound to form homocysteine. Finally, homocysteine is quantified using Ellman's reagent and the accompanying absorption change at 412nm through recording using a microplate format. Notably, SAMT and most AdoMet-dependent methyltransferases undergo marked AdoHcy-mediated product inhibition. As such, an additional advantage of this assay which includes AdoHcy nucleosidase is the destruction of AdoHcy, thus alleviating product inhibition. Under our assay conditions, complete substrate conversion is observed and precise kinetic parameters can be determined in a facile and quantitative manner. This assay should be generally applicable to other AdoMet-dependent methyltransferases. Moreover, the procedure is easily amendable to batch assay and high-throughput screening approaches.
我们在此报告一种用于水杨酸羧基甲基转移酶（SAMT）的酶联比色测定法，该方法利用S-腺苷-L-甲硫氨酸（AdoMet或SAM）作为甲基供体。在该测定中，AdoMet依赖性转甲基反应的常见产物S-腺苷-L-高半胱氨酸（AdoHcy或SAH）首先被重组AdoHcy核苷酶（EC 3.2.2.9）水解为腺嘌呤和S-核糖基高半胱氨酸。重组LuxS（S-核糖基高半胱氨酸酶，EC 3.2.1.148）切割后一种化合物形成高半胱氨酸。最后，使用Ellman试剂对高半胱氨酸进行定量，并通过微孔板形式记录在412nm处伴随的吸光度变化。值得注意的是，SAMT和大多数AdoMet依赖性甲基转移酶会受到明显的AdoHcy介导的产物抑制。因此，该包含AdoHcy核苷酶的测定法的另一个优点是AdoHcy的破坏，从而减轻产物抑制。在我们的测定条件下，观察到底物完全转化，并且可以以简便且定量的方式确定精确的动力学参数。该测定法通常应适用于其他AdoMet依赖性甲基转移酶。此外，该程序易于修改以适用于批量测定和高通量筛选方法。
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