Localization of dihydroorotate oxidase in myocardium and kidney cortex of the rat. An electron microscopic study using the cerium technique.
Biochemical studies have demonstrated that dihydroorotate dehydrogenase (DHOdehase; EC 1.3.3.1 or 1.3.99.11) is the sole enzyme of de novo pyrimidine synthesis in mitochondria, whereas the rest of the pathway takes place in the cytosol. The dehydrogenation of dihydroorotate to orotate is linked to the respiratory chain via ubiquinone. In this study, we show for the first time the ultrastructural localization of DHOdehase. Since the purified enzyme was found to act both as dehydrogenase and as oxidase, the cerium capture technique for detecting enzymatically generated hydrogen peroxide could be applied to pin-point the in situ activity of DHOdehase oxidase in mitochondria of rat heart and kidney cortex. Cerium perhydroxide as the final reaction product was detected predominantly in the matrix with some focal condensation along the inner membrane, but not in the intermembrane space. From this pattern of localization, it is concluded that the active site of the membrane-bound enzyme could face the mitochondrial matrix similar to succinate dehydrogenase. The reliability of the applied method for the demonstration of DHOdehase oxidase was demonstrated by the addition of Brequinar sodium to the incubation medium. This quinoline-carboxylic acid derivative is a potent inhibitor of DHOdehase and has proven anti-proliferative activity. The present observations do not ascertain whether the oxidase is permanently active as a constant portion of the enzyme in vivo, similar to xanthine oxidase/dehydrogenase. However, DHOdehase should be considered as a source of radical oxygen species under pathophysiological conditions.
Angermüller
S
S
Department of Anatomy and Cell Biology II, University of Heidelberg, Germany.
Löffler
M
M
D000818:Animals; D002451:Cell Compartmentation; D004081:Dihydroorotate Oxidase / Q000032:analysis*; D007672:Kidney Cortex / Q000201:enzymology* / Q000648:ultrastructure; D008297:Male; D008854:Microscopy, Electron; D008929:Mitochondria, Heart / Q000201:enzymology / Q000648:ultrastructure; D009206:Myocardium / Q000201:enzymology*; D051381:Rats; D017207:Rats, Sprague-Dawley
D004081:Dihydroorotate Oxidase