Abstract
Nitrate is an important source of nitrogen for ectomycorrhizal fungi and their hosts. Nitrate reductase catalyses the first step in the assimilation of nitrate into organic nitrogen and has not yet been successfully characterized from a mycorrhizal ascomycete. The extraction and properties of NR from the mycorrhizal Wilcoxina mikolae var. mikolae are described. The strategy adopted for the extraction included the development of culture conditions that were amenable to de-repression and induction of NR, as well as considerations of the lability of the enzyme. Growth on a neutral nitrogen source, the use of an artificial electron donor and rapid partial purification in the presence of phenyl methyl sulfonyl fluoride (PMSF) contributed to the extraction of a fully functional NADPH-specific NR from Wilcoxina . The optimum pH for nitrate reduction by this enzyme was 7·0 in 100 m m phosphate. The apparent K m for NADPH was 35 m and the K d for nitrate was 200 m . The functional 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 endogenous factors during extraction.
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