Biochimica et Biophysica Acta (BBA) - General Subjects ( IF 3 ) Pub Date : 2020-10-22 , DOI: 10.1016/j.bbagen.2020.129767 Xavier Serfaty , Pauline Lefrançois , Chantal Houée-Levin , Stéphane Arbault , Laura Baciou , Tania Bizouarn
Background
The production of superoxide anions (O2•-) by the phagocyte NADPH oxidase complex has a crucial role in the destruction of pathogens in innate immunity. Majority of in vitro studies on the functioning of NADPH oxidase indirectly follows the enzymatic reaction by the superoxide reduction of cytochrome c (cyt c). Only few reports mention the alternative approach consisting in measuring the NADPH consumption rate.
When using membrane vesicles of human neutrophils, the enzyme specific activity is generally found twice higher by monitoring the NADPH oxidation than by measuring the cyt c reduction. Up to now, the literature provides only little explanations about such discrepancy despite the critical importance to quantify the exact enzyme activity.
Methods
We deciphered the reasons of this disparity in studying the role of key parameters, including.
cyt c and arachidonic acid concentrations, in conjunction with an ionophore, a detergent and using Clark electrode to measure the O2 consumption rates.
Results
Our results show that the O2•- low permeability of the vesicle membrane as well as secondary reactions (O2•- and H2O2 disproportionations) are strong clues to shed light on this inconsistency.
Conclusion and General Significance
These results altogether indicate that the cyt c reduction method underestimates the accurate Nox2 activity.
中文翻译:
囊泡环境对体外Nox2活性测量的影响
背景
吞噬细胞NADPH氧化酶复合物产生的超氧阴离子(O 2 •-)在破坏先天性免疫中的病原体方面起着至关重要的作用。大部分有关NADPH氧化酶功能的体外研究都是通过细胞色素c(cyt c)的超氧化物还原而间接引起的酶促反应。只有很少的报告提到替代方法,即测量NADPH消耗率。
当使用人嗜中性白细胞的膜囊泡时,通常通过监测NADPH氧化发现酶比活性比通过测量cyt c还原率高两倍。到目前为止,尽管量化精确的酶活性至关重要,但文献仅对这种差异提供了很少的解释。
方法
我们在研究关键参数的作用时,包括了这种差异的原因,包括。
cyt c和花生四烯酸的浓度,结合离子载体,去污剂并使用Clark电极测量O 2消耗速率。
结果
我们的结果表明,囊膜的O 2 •-低渗透性以及次级反应(O 2 •-和H 2 O 2歧化)是阐明这种矛盾的有力线索。
结论与一般意义
这些结果完全表明,cyt c还原方法低估了准确的Nox2活性。