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叫我阿離哈92新蟲 (初入文壇)
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[求助]
關(guān)于酶活性檢測(cè)的問(wèn)題 已有1人參與
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關(guān)于酶的檢測(cè)的，文獻(xiàn)中說(shuō)到的NADPH-generating system 是怎么參與到酶檢測(cè)的，起到的作用是什么？這段就沒(méi)怎么看懂，求能人指教 The incubation mixture, with a total volume of 200 μl, consisted of 100mM potassium phosphate buffer (pH 7.4), NADPH-generating system (1 mM NADP+, 10 mM glucose-6-phosphate, 1 unit/mL of glucose-6-phosphate dehydrogenase, and 4 mM MgCl2), and human liver microsomes or CYPs. In all experiments, NCMN (20 mM dissolved in acetonitrile previously) was serially diluted to the required concentrations and the final concentration of acetonitrile did not exceed 1% (v/v) in the mixture. After preincubation at 37oC for 3 min, the reaction was initiated by adding NADPH-generating system and further incubated at 37oC in a shaking water bath. The reaction was terminated by the addition of ice-cold acetonitrile (100 μl). The mixture was kept on ice until it was centrifuged at 20,000×g for 20 min at 4oC. Aliquots of supernatants were stored at -20oC until analysis. Aliquots of supernatants were taken for further fluorescence analysis (Gain = 60 for substrate and Gain =100 for metabolite). Control incubations without NADPH-generating system or without substrate or without CYP enzyme sources were carried out to investigate whether the formation of metabolite(s) was enzyme (CYP)- and NADPH-dependent. All incubations throughout the study were carried out in duplicate with SD.values generally below 10%. |
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cai3110274
木蟲 (職業(yè)作家)
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【答案】應(yīng)助回帖
★ ★ ★ ★ ★
感謝參與，應(yīng)助指數(shù) +1
叫我阿離哈92: 金幣+5, ★有幫助 2017-05-13 21:33:29
感謝參與，應(yīng)助指數(shù) +1
叫我阿離哈92: 金幣+5, ★有幫助 2017-05-13 21:33:29
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NADPH-generating system (1 mM NADP+, 10 mM glucose-6-phosphate, 1 unit/mL of glucose-6-phosphate dehydrogenase, and 4 mM MgCl2） 這個(gè)是NADPH再生體系，是PPP途徑的第一步反應(yīng)，看下反應(yīng)式就知道了 CYP酶反應(yīng)時(shí)消耗NADPH，再生體系保證了NADPH的供應(yīng) 大概是這么個(gè)意思 |
2樓2017-04-28 08:23:25
叫我阿離哈92
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3樓2017-04-28 15:53:18
cai3110274
木蟲 (職業(yè)作家)
- 應(yīng)助: 29 (小學(xué)生)
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4樓2017-05-13 23:15:22
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