Duquette P H, Holtzman J L
J Pharmacol Exp Ther. 1979 Oct;211(1):213-8.
A sensitive and convenient radioassay for the in vitro determination of ethylmorphine N-demethylase and O-de-ethylase activity has been developed. Ethylmorphine[6-3H] was prepared by reduction of the corresponding morphinone in nearly quantitative yield. After incubation with hepatic microsomes from male rats, the reaction was terminated by the addition of 5 ml of acetone. The sample was saturated with potassium acetate and extracted twice with acetone giving complete extraction of the radiolabeled ethylmorphine and its metabolites. After the combined organic phases were evaporated, the samples were dissolved in methanol and applied to a Silica Gel GF plate with subsequent development in ethyl acetate-methanol-concentrated NH4OH. The amount of radioactivity detected for the morphine and norethylmorphine bands at zero time was approximately 0.05% of the original amount of labeled ethylmorphine added to the incubation media. Similarly, the Km values were 52 and 250 microns for the O- and N-dealkylation respectively, while the Vmax values were 5.0 and 1.8 nmol/mg of protein per min. Finally, with this assay we have observed constant specific activity for both the N- and O-dealkylation of ethylmorphine[6-3H] with as little as 10 micrograms of microsomal protein per ml of incubation media.
已开发出一种灵敏且便捷的放射分析法，用于体外测定乙基吗啡N-脱甲基酶和O-脱乙基酶的活性。通过还原相应的吗啡酮，以近乎定量的产率制备了[6-³H]乙基吗啡。与雄性大鼠的肝微粒体孵育后，加入5毫升丙酮终止反应。样品用醋酸钾饱和，并用丙酮萃取两次，从而使放射性标记的乙基吗啡及其代谢产物完全萃取出来。合并的有机相蒸发后，将样品溶解在甲醇中，点样于硅胶GF板上，随后在乙酸乙酯-甲醇-浓氨水展开剂中展开。在零时间检测到的吗啡和去甲乙基吗啡条带的放射性约为添加到孵育介质中的标记乙基吗啡原始量的0.05%。同样，O-脱烷基化和N-脱烷基化的Km值分别为52和250微摩尔，而Vmax值分别为每分钟每毫克蛋白质5.0和1.8纳摩尔。最后，使用该分析法，我们观察到，每毫升孵育介质中仅含10微克微粒体蛋白时，[6-³H]乙基吗啡的N-脱烷基化和O-脱烷基化的比活性均保持恒定。
