The Regulation of Nitrate Reductase in the Fungus Aspergillus nidulans

Abstract

Nitrate reductase induction by nitrate and repression by NH,+ has been studied extensively in the simple eukaryote Aspergillus nidulans (Pateman & Cove, 1967; Cove & Pateman, 1968). Wild-type cells grown on minimal medium, with 20m~-NaNO~ as the nitrogen source, rapidly lose nitrate reductase activity when transferred either to minimal medium without N (-N medium) or to minimal medium without C and N (-CN medium) or to -CN medium plus 100m-L-glutamate as the sole N and C source. Hynes (1973), whose work we have confirmed, found that transferring pre-grown cells from 10mM-NaN03 to a medium lacking a C source (-C medium) resulted in the greatest decrease in activity, even in the presence of 10m-NaN03. Cyclohexamide, an inhibitor of protein synthesis at a concentration of lOpg/ml, did not prevent the loss of activity. When cell-free extracts of wild-type cells, grown on -N medium+20m~-NaNO~, were centrifuged at 6OOOOg for 20min and incubated for periods of up to 1 h with 2.65m1 of orthophosphate buffer, pH7.75,+3.5pg of FAD, there was a rapid loss of nitrate reductase activity (see Fig. 1). There was a similar loss of enzyme activity when either 1lOm-NaNO3 or 35m-NH was,+ added to the incubation mixture. When NADH-linked nitrate reductase extracts from Nitrobacter agilis, pre-grown on nitrate, are incubated in the absence of nitrate, rapid inactivation of the enzyme results. Herrera & Nicholas (1974) found that this inactivation was the result of over-reduction with NADH. Several workers also found that nitrate reductase is inactivated by reduced