Respiratory chain components involved in the glycerophosphate dehydrogenase-dependent ROS production by brown adipose tissue mitochondria |
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Authors: | Marek Vrbacký Tomáš Mrá?ek Pavel Ješina Josef Houštěk |
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Affiliation: | a Department of Bioenergetics, Institute of Physiology, Academy of Sciences of the Czech Republic, Vídeňská 1083, CZ 142 20 Prague, Czech Republic b Institute of Inorganic Chemistry, Academy of Sciences of the Czech Republic, 250 68 ?e?, Czech Republic |
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Abstract: | Involvement of mammalian mitochondrial glycerophosphate dehydrogenase (mGPDH, EC 1.1.99.5) in reactive oxygen species (ROS) generation was studied in brown adipose tissue mitochondria by different spectroscopic techniques. Spectrofluorometry using ROS-sensitive probes CM-H2DCFDA and Amplex Red was used to determine the glycerophosphate- or succinate-dependent ROS production in mitochondria supplemented with respiratory chain inhibitors antimycin A and myxothiazol. In case of glycerophosphate oxidation, most of the ROS originated directly from mGPDH and coenzyme Q while complex III was a typical site of ROS production in succinate oxidation. Glycerophosphate-dependent ROS production monitored by KCN-insensitive oxygen consumption was highly activated by one-electron acceptor ferricyanide, whereas succinate-dependent ROS production was unaffected. In addition, superoxide anion radical was detected as a mGPDH-related primary ROS species by fluorescent probe dihydroethidium, as well as by electron paramagnetic resonance (EPR) spectroscopy with DMPO spin trap. Altogether, the data obtained demonstrate pronounced differences in the mechanism of ROS production originating from oxidation of glycerophosphate and succinate indicating that electron transfer from mGPDH to coenzyme Q is highly prone to electron leak and superoxide generation. |
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Keywords: | BAT brown adipose tissue CM-H2DCFDA 5-(and-6)-chloromethyl-2 7-dichlorodihydrofluorescein diacetate acetyl ester DCPIP 2 6-dichlorophenolindophenol DHE dihydroethidium DMPO 5 5-dimethyl-1-pyrroline-N-oxide mGPDH mitochondrial FAD-dependent glycerophosphate dehydrogenase SDH succinate dehydrogenase ROS reactive oxygen species |
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