Valdez Laura B, Zaobornyj Tamara, Boveris Alberto
Laboratory of Free Radical Biology and Pharmacology, School of Pharmacy and Biochemistry, University of Buenos Aires, Brazil.
Methods Enzymol. 2005;396:444-55. doi: 10.1016/S0076-6879(05)96037-0.
The functional activity of mitochondrial nitric oxide synthase (mtNOS) is determined by inhibiting O2 uptake and by enhancing H2O2 production. The effect of mtNOS activity on mitochondrial O2 uptake is assayed in state 3 respiration in two limit conditions of intramitochondrial NO: at its maximal and minimal levels. The first condition is achieved by supplementation with L-arginine and superoxide dismutase (SOD), and the second by addition of an NOS inhibitor and oxyhemoglobin. The difference between state 3 O2 uptake in both conditions constitutes the mtNOS functional activity in the inhibition of cytochrome oxidase activity. The functional activity of mtNOS in enhancing mitochondrial H2O2 generation in state 4 is given by the NO inhibition of ubiquinol-cytochrome c reductase activity. Simple determinations with the oxygen electrode or the measurement of mitochondrial H2O2 production can be used to assay the effects of physiological and pharmacological treatments on mtNOS activity.
线粒体一氧化氮合酶（mtNOS）的功能活性通过抑制氧气摄取和增强过氧化氢生成来确定。在两种线粒体内一氧化氮（NO）的极限条件下，即在其最大和最小水平时，通过测定状态3呼吸中mtNOS活性对线粒体氧气摄取的影响。第一种条件是通过补充L-精氨酸和超氧化物歧化酶（SOD）来实现，第二种条件是通过添加一氧化氮合酶抑制剂和氧合血红蛋白来实现。两种条件下状态3氧气摄取的差异构成了mtNOS在抑制细胞色素氧化酶活性方面的功能活性。mtNOS在增强状态4线粒体过氧化氢生成方面的功能活性由NO对泛醌-细胞色素c还原酶活性的抑制作用给出。使用氧电极进行简单测定或测量线粒体过氧化氢生成可用于检测生理和药物处理对mtNOS活性的影响。
