DIRECT MEASUREMENT OF NAD(P)H - QUINONE REDUCTASE FROM CELLS CULTURED IN MICROTITER WELLS - A SCREENING ASSAY FOR ANTICARCINOGENIC ENZYME INDUCERS
DIRECT MEASUREMENT OF NAD(P)H - QUINONE REDUCTASE FROM CELLS CULTURED IN MICROTITER WELLS - A SCREENING ASSAY FOR ANTICARCINOGENIC ENZYME INDUCERS
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DOI：
10.1016/0003-2697(88)90292-8
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发表时间：
1988-03-01
影响因子：
2.9
通讯作者：
SANTAMARIA, AB
中科院分区：
文献类型：
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作者：
PROCHASKA, HJ;SANTAMARIA, AB
我们描述了一种在培养细胞中快速直接测定NAD(P)H（醌受体）氧化还原酶（EC 1.6.99.2）活性的方法，该方法适合于鉴定和纯化该解毒酶的诱导剂。将Hepa 1c1c7小鼠肝癌细胞置于96孔微滴板中，培养24小时，再暴露于诱导剂中24小时。然后将细胞裂解，并通过添加含有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]的反应混合物来测定醌还原酶活性。醌还原酶通过NADPH催化menadione还原为menadiol， MTT被menadiol非酶还原，形成蓝色，可以在微滴板吸光度仪上定量。该反应90%以上为双酚抑制，且依赖于甲萘醌。所得结果与从较大的板上收集细胞、制备细胞溶胶和进行分光光度测量所得的结果相当。
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 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, preparting cytosols, and carrying out spectrophotometric measurements.