Enzymhistochemischer und immunhistochemischer Nachweis der Dihydroorotat-Dehydrogenase im ZSN

The fourth enzyme of the de novo pyrimidine biosynthesis, dihydroorotate dehydrogenase, catalyzes the oxidation of dihydroorotate to orotate. Thus, DHODH is decisively involved in the organism’s supply with uridinemonophosphate, as well as cytidine and thymidine nucleotides. Representing essential elements of DNA and RNA, these molecules play an important role in cell metabolism. In the present work, an extensive presentation of DHODH in rat’s CNS was realized for the first time. This study gave information about the localization and activity of DHODH in different areas of rodent brain. First of all the enzyme was detected via SDS polyacrylamide gel electrophoresis, followed by several Western blots. As a precondition for these tests, the antibody titres were experimentally optimized. Subsequently, a protein preparation and detection by the use of the Bradford protein assay was made in CNS tissue. The subsequent SDS-PAGE revealed the existence of DHODH in all dissected tissues. In detail the enzyme was detected in the brain areas below: cortex, striatum, hippocampus, thalamus, hypothalamus, superior colliculi, cerebellum, spinal cord, brainstem and inferior colliculi. Only the extracts of brainstem and inferior colliculi showed bands of low intensity. The enzyme activity of DHODH (oxidation of dihydroorotate to orotate) in rat brain, was investigated by the nitroblue tetrazolium (NBT) assay. High activity appeared in brain areas, which are relatively rich in neurons. Furthermore, grey matter of the spinal chord as well the plexus choroideus showed the subcellular location of DHODH. Thus, enzyme activity of DHODH in rat brain was proved, that supports the theory of an active pyrimdine de novo synthesis taking place in brain. Using the NBT assay, the effect of different inhibitors on DHODH activity was investigated in rat brain. Established inhibitors, such as DCL, A77 1726, atovaquone and redoxal, turned out to be especially potent. An inhibition of rat DHODH by brequinar was also shown, even though to a minor extent. Furthermore, the inhibitory effect of the barely reviewed substance UID1 on DHODH was presented. Besides, UID1 was capable of inhibiting succinate dehydrogenase activity in rat brain. Finally, the localization of DHODH on cellular level was defined by the use of immunohistochemical methods in selected brain areas. Using an affinity-purified antibody, the findings of the diverse activity tests were confirmed, suggesting a neuronal enzyme localization. Concluding it has to be stated, that DHODH is active in CNS of adult rats and that it is (with the exception of plexus choroideus cells) predominantly localized in neurons. An important role of dihydroorotate-dehydrogenase in rat brain seems to be possible.