Beaudouin C, Haurat G, Fraisse L, Souppe J, Renaud B
Laboratoire de Neuropharmacologie-CNRS UMR 105, Faculté de Pharmacie, Université Claude Bernard, Lyon, France.
J Chromatogr. 1993 Mar 5;613(1):51-8. doi: 10.1016/0378-4347(93)80196-b.
A simple and rapid method for measuring phenylethanolamine N-methyltransferase (PNMT) activity by high-performance liquid chromatography (HPLC) with ultraviolet (UV) detection is described. This assay requires a partially purified PNMT preparation derived from bovine adrenals, with noradrenaline and S-adenosyl-L-methionine (SAM) as co-substrates. After incubation, the reaction is stopped by addition of acid and the reaction mixture is analysed directly by HPLC. The enzymatically formed S-adenosyl-L-homocysteine (SAH) is detected at 258 nm and determined. Under optimum conditions, the stability of SAH allowed automation of the HPLC detection. This assay was validated by the determination of the kinetic properties of PNMT. Km values for noradrenaline and SAM defined in this assay (16 and 5.7 microM, respectively) are consistent with previously published values. This assay is simple enough to be used for large series of measurements of PNMT activity testing new methyl acceptors, potential inhibitors or PNMT activity in adrenal medulla.
本文描述了一种通过高效液相色谱（HPLC）结合紫外（UV）检测来测定苯乙醇胺N-甲基转移酶（PNMT）活性的简单快速方法。该测定需要一种源自牛肾上腺的部分纯化的PNMT制剂，以去甲肾上腺素和S-腺苷-L-甲硫氨酸（SAM）作为共底物。孵育后，通过加入酸终止反应，反应混合物直接通过HPLC进行分析。在258nm处检测并测定酶促形成的S-腺苷-L-高半胱氨酸（SAH）。在最佳条件下，SAH的稳定性使HPLC检测能够自动化。通过测定PNMT的动力学性质对该测定进行了验证。该测定中确定的去甲肾上腺素和SAM的Km值（分别为16和5.7μM）与先前发表的值一致。该测定足够简单，可用于对新的甲基受体、潜在抑制剂或肾上腺髓质中PNMT活性进行大量的PNMT活性测试测量。
