Dihydroorotate dehydrogenase (EC 1.3.3.1 or EC 1.3.99.11) catalyzes the fourth sequential step in the de novo synthesis of
uridine monophosphate. In eukaryotes it is located in the inner mitochondrial membrane, with
ubiquinone as the proximal and
cytochrome oxidase as the ultimate electron transfer system, whereas the rest of
pyrimidine biosynthesis takes place in the cytosol. Here, the distribution of
dihydroorotate dehydrogenase activity in cryostat sections of various rat tissues, and tissue samples of human skin and kidney, was visualized by light microscopy using the
nitroblue tetrazolium technique. In addition, a
hydrogen peroxide-producing
oxidase side-reactivity of
dihydroorotate dehydrogenase could be visualized by trapping the
peroxide with
cerium-diaminobenzidine. The pattern of activity was similar to that of
succinate dehydrogenase, but revealed a less intensive staining. High activities of
dihydroorotate dehydrogenase were found in tissues with known proliferative, regenerative, absorptive or excretory activities, e.g., mucosal cells of the ileum and colon crypts in the gastrointestinal tract, cultured
Ehrlich ascites tumor cells, and proximal tubules of the kidney cortex, whilst lower activities were present in the periportal area of the liver, testis and spermatozoa, prostate and other glands, and skeletal muscle.
Dihydroorotate dehydrogenase and
succinate dehydrogenase activity in
Ehrlich ascites tumor cells grown in
suspension culture were quantified by application of
nitroblue tetrazolium or cyanotolyl tetrazolium and subsequent extraction of the insoluble
formazans with organic
solvents. The ratio of
dihydroorotate dehydrogenase to
succinate dehydrogenase activity was 1:4. This was in accordance with that of 1:5 obtained from oxygen consumption measurement of isolated mitochondria on addition of
dihydroorotate or
succinate. The ratio determined with mitochondria from animal tissues was up to 1:15 (rat liver, bovine heart). The application of the
enzyme inhibitors brequinar sodium and
toltrazuril verified the specificity of the histochemical and biochemical methods applied.