A study of the stability and properties of nitrate reductase from the ectomycorrhizal fungus Pisolithus tinctorius

A study of the stability and properties of nitrate reductase from the ectom ycorrhizal Fungus Pisolithus tinctorius. The ectomycorrhizal basidiomycete Pisolithus tinctorius grew better in the presence of nitrate than on ammoni um-supplemented media. Nitrate utilization led to an increase of pH, wherea s 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 Pisolithu s tinctorius was studied in order to obtain the most stable activity, it co nsisted of 100 mM K-phosphate buffer, pH 7.5 including 1 mM EDTA, 10 muM FA D, 1 muM Na molybdate, 1 % casein and 1 % fresh weight polyvinylpolypyrroli done. Despite the use of these protectants, nitrate reductase partially pur ified by ammonium sulfate fractionation and anion-exchange chromatography o n DEAE-Trisacryl proved to be very unstable. Thus at -25 degreesC, the acti vity was reduced by half within 4 weeks, and at 4 degreesC, 60 % of the act ivity disappeared within 4 hours. At 20 degreesC the activity was completel y lost in less than 4 hours. The optimal pH value for the reduction of nitr ate was 7.5. The enzyme was specific for NADPH and the Michaelis constants for this cofactor and nitrate were 49.6 muM and 328 muM, respectively. Nitr ate reductase activity was induced in the presence of nitrate and repressed by ammonium. Transfer experiments of colonies from ammonium- to nitrate-co ntaining media, and vice versa, revealed that enzyme activity appeared with in a few minutes, whereas disappearance of the activity took several hours, half of the activity being lost within 20 hours. (C) 2000 Adac/Editions sc ientifiques et medicales Elsevier SAS.