PROPERTIES OF NITRATE REDUCTASE FROM THE MYCORRHIZAL ASCOMYCETE WILCOXINA-MIKOLAE VAR MIKOLAE

Nitrate is an important source of nitrogen for ectomycorrhizal fungi a nd their hosts. Nitrate reductase catalyses the first step in the assi milation of nitrate into organic nitrogen and has not yet been success fully characterized from a mycorrhizal ascomycete. The extraction and properties of NR from the mycorrhizal Wilcoxina mikolae var. mikolae a re described. The strategy adopted for the extraction included the dev elopment of culture conditions that were amenable to de-repression and induction of NR, as well as considerations of the lability of the enz yme. Growth on a neutral nitrogen source, the use of an artificial ele ctron donor and rapid partial purification in the presence of phenyl m ethyl sulfonyl fluoride (PMSF) contributed to the extraction of a full y functional NADPH-specific NR from Wilcoxina. The optimum pH for nitr ate reduction by this enzyme was 7.0 in 100 mM phosphate. The apparent K-m for NADPH was 35 mu M and the K-d for nitrate was 200 mu M. The f unctional properties of this enzyme are similar to those reported for other filamentous fungi. The NR from Wilcoxina is thought to be highly prone to nitrogen metabolite repression, and inactivation by endogeno us factors during extraction.