Measurement of Iso-orotate Decarboxylase activity in Neurospora Crassa, /

Abstract

The biosynthesis of uridine-5' -monophosphate (UMP) can occur through two pathways. The first and most common is the de novo pathway, while the second and more unique pathway is the thymidine salvage pathway. The fungus Neurospora crassa, unlike many organisms, contains both but utilizes the second of these pathways to convert thymidine to uracil. My research focuses on the fourth enzyme of the thymidine salvage pathway, iso-orotate decarboxylase. Preliminary characterization of IDCase was determined by a radioactivity based assay using 14 C to label the substrate lOA. This decarboxylation assay measures the amount of IDCase activity by detecting the amount of radio-labeled CO2 released from lOA in the reaction at a specified time point. Wild type N. crassa strains had the highest specific enzyme activity of 2.5 nmol/min/mg when grown in a shake culture for 18 hours at 30°C. We have tested iso-orotate decarboxylase levels at various time points in the N. crassa life cycle. We have also examined the rate of induction of IDCase activity in the presence of nitrate as well as in the N. crassa strains containing the 2TI:1 and nit-2 mutations. As uracil has been shown to be an inducer of the IDCase levels and may therefore act as an inducer for the thymidine salvage pathway.