Functional activity of mitochondrial nitric oxide synthase

VALDEZ LB; ZAOBORNYJ T; BOVERIS A.

Methods Enzymol

Año: 2005 vol. 396 p. 444 - 455

The functional activity of mitochondrial nitric oxide synthase (mtNOS) is determined both in inhibiting O2 uptake and in enhancing H2O2 production. The effect of mtNOS activity on mitochondrial O2 uptake is assayed in state 3 respiration. Respiratory rates are determined in two limit conditions of intramitochondrial NO: at its maximal and minimal levels. The first condition is achieved by supplementation with L-arginine and SOD, and the second by addition of NOS inhibitor and of oxyhemoglobin. The difference between the state 3 O2 uptake with L-arginine and SOD and with NOS inhibitor and oxyhemoglobin indicates the mtNOS functional activity in the inhibition of cytochrome oxidase activity. The regulatory activity of mtNOS on mitochondrial H2O2 generation is assayed in mitochondrial state 4. The effects on H2O2 production are  explained by the NO inhibition of ubiquinol-succinate-cytochrome c reductase activity that enhances H2O2 production. Simple respiratory determinations with the oxygen electrode or through the measurement of mitochondrial H2O2 production can be used to assay effects of physiological or pharmacological treatments on mtNOS activity.