Étude de la stabilité et de quelques propriétés de la nitrate réductase du champignon ectomycorhizien Pisolithus tinctorius

Abstract

A study of the stability and properties of nitrate reductase from the ectomycorrhizal fungus Pisolithus tinctorius. The ectomycorrhizal basidiomycete Pisolithus tinctorius grew better in the presence of nitrate than on ammonium-supplemented media. Nitrate utilization led to an increase of pH, whereas ammonium utilization was accompanied by a pH drop. The composition of the buffer for extraction of the nitrate reductase (EC 1.6.6.3) from Pisolithus tinctorius was studied in order to obtain the most stable activity. It consisted of 100 mM K-phosphate buffer, pH 7.5 including 1 mM EDTA, 10 μM FAD, 1 μM Na molybdate, 1 % casein and 1 % fresh weight polyvinylpolypyrrolidone. Despite the use of these protectants, nitrate reductase partially purified by ammonium sulfate fractionation and anion-exchange chromatography on DEAE-Trisacryl proved to be very unstable. Thus at -25 °C, the activity was reduced by half within 4 weeks, and at 4 °C, 60 % of the activity disappeared within 4 hours. At 20 °C the activity was completely lost in less than 4 hours. The optimal pH value for the reduction of nitrate was 7.5. The enzyme was specific for NADPH and the Michaelis constants for this cofactor and nitrate were 49.6 μM and 328 μM, respectively. Nitrate reductase activity was induced in the presence of nitrate and repressed by ammonium. Transfer experiments of colonies from ammonium- to nitrate-containing media, and vice versa, revealed that enzyme activity appeared within a few minutes, whereas disappearance of the activity took several hours, half of the activity being lost within 20 hours.