CATALYTIC ENZYME-HISTOCHEMISTRY AND BIOCHEMICAL-ANALYSIS OF DIHYDROOROTATE DEHYDROGENASE OXIDASE AND SUCCINATE-DEHYDROGENASE IN MAMMALIAN-TISSUES, CELLS AND MITOCHONDRIA

Dihydroorotate dehydrogenase (EC 1.3.3.1 or EC 1.3.99.11) catalyzes th e fourth sequential step in the de novo synthesis of uridine monophosp hate. In eukaryotes it is located in the inner mitochondrial membrane, with ubiquinone as the proximal and cytochrome oxidase as the ultimat e electron transfer system, whereas the rest of pyrimidine biosynthesi s takes place in the cytosol. Here, the distribution of dihydroorotate dehydrogenase activity in cryostat sections of various rat tissues, a nd tissue samples of human skin and kidney, was visualized by light mi croscopy using the nitroblue tetrazolium technique. In addition, a hyd rogen peroxide-producing oxidase side-reactivity of dihydroorotate deh ydrogenase could be visualized by trapping the peroxide with cerium-di aminobenzidine. The pattern of activity was similar to that of succina te dehydrogenase, but revealed a less intensive staining. High activit ies of dihydroorotate dehydrogenase were found in tissues with known p roliferative, regenerative, absorptive or excretory activities, e.g., mucosal cells of the ileum and colon crypts in the gastrointestinal tr act, 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 dehyd rogenase activity in Ehrlich ascites tumor cells grown in suspension c ulture were quantified by application of nitroblue tetrazolium or cyan otolyl tetrazolium and subsequent extraction of the insoluble formazan s 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 m itochondria on addition of dihydroorotate or succinate. The ratio dete rmined with mitochondria from animal tissues was up to 1:15 (rat liver , bovine heart). The application of the enzyme inhibitors brequinar so dium and toltrazuril verified the specificity of the histochemical and biochemical methods applied.