Prochaska H J, Santamaria A B
Department of Pharmacology and Molecular Sciences, Johns Hopkins University, School of Medicine, Baltimore, Maryland 21205.
Anal Biochem. 1988 Mar;169(2):328-36. doi: 10.1016/0003-2697(88)90292-8.
We describe a rapid and direct assay of NAD(P)H:(quinone-acceptor) oxidoreductase (EC 1.6.99.2) activity in cultured cells suitable for identifying and purifying inducers of this detoxication enzyme. Hepa 1c1c7 murine hepatoma cells are plated in 96-well microtiter plates, grown for 24 h, and exposed to inducing agents for another 24 h. The cells are then lysed and quinone reductase activity is assayed by the addition of a reaction mixture containing an NADPH-generating system, menadione (2-methyl-1,4-naphthoquinone), and MTT [3-(4,-5-dimethylthiazo-2-yl)-2,5-diphenyltetrazolium bromide]. Quinone reductase catalyzes the reduction of menadione to menadiol by NADPH, and MTT is reduced nonenzymatically by menadiol resulting in the formation of a blue color which can be quantitated on a microtiter plate absorbance reader. The reaction is more than 90% dicoumarol inhibitable and menadione dependent. The results are comparable to those obtained by harvesting cells from larger plates, preparing cytosols, and carrying out spectrophotometric measurements.
我们描述了一种适用于鉴定和纯化这种解毒酶诱导剂的、在培养细胞中快速直接检测NAD(P)H:(醌-受体)氧化还原酶(EC 1.6.99.2)活性的方法。将Hepa 1c1c7小鼠肝癌细胞接种于96孔微量滴定板中，培养24小时，再暴露于诱导剂中24小时。然后裂解细胞，通过加入含有NADPH生成系统、甲萘醌(2-甲基-1,4-萘醌)和MTT [3-(4,5-二甲基噻唑-2-基)-2,5-二苯基溴化四唑]的反应混合物来检测醌还原酶活性。醌还原酶催化甲萘醌被NADPH还原为甲萘二酚，甲萘二酚非酶促还原MTT，导致形成蓝色，可在微量滴定板吸光读数仪上进行定量。该反应超过90%可被双香豆素抑制且依赖于甲萘醌。结果与从较大平板收获细胞、制备胞质溶胶并进行分光光度测量所获得的结果相当。
