We examined whether root mitochondria and mitochondrial membranes produce nitric oxide (NO) exclusively by reduction of nitrite or also via a nitric oxide synthase (NOS), and to what extent direct NO measurements could become undetectable due to NO oxidation. Chemiluminescence detection of NO in the gas phase was used to monitor NO emission from suspensions (i.e. direct chemiluminescence). For comparison, diaminofluorescein (DAF) and diaminorhodamine (DAR) were used as NO indicators. NO oxidation to nitrite and nitrate was quantified after reduction of nitrite + nitrate to NO by vandium (III) with subsequent chemiluminescence detection (i.e. indirect chemiluminescence). Nitrite and NADH consumption were also measured. Anaerobic nitrite-dependent NO emission was exclusively associated with the membrane fraction, without participation of matrix components. Rates of nitrite and NADH consumption matched, whereas the rate of NO emission was lower. In air, mitochondria apparently produced no nitrite-dependent NO, and no NOS activity was detected by direct or indirect chemiluminescence. In contrast, with DAF-2 or DAR-4M, an l-arginine-dependent fluorescence increase took place. However, the response of this apparent low NOS activity to inhibitors, substrates and cofactors was untypical when compared with commercial inducible NOS (iNOS), and the existence of NOS in root mitochondria is therefore doubtful. In a solution of commercial iNOS, about two-thirds of the NO (measured as NADPH consumption) were oxidized to nitrite + nitrate. Addition of mitochondria to iNOS decreased the apparent NO emission, but without a concomitant increase in nitrite + nitrate formation. Thus, mitochondria appeared to accelerate oxidation of NO to volatile intermediates.
Keywords: Chemiluminescence; Fluorescence; Mitochondria; Nitric oxide; Nitric oxide synthase; Nitrite
Journal Article. 5067 words. Illustrated.
Subjects: Biochemistry ; Molecular and Cell Biology ; Plant Sciences and Forestry
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