Optimum conditions for cotton nitrate reductase extraction and activity measurement

Conditions of nitrate reductase extraction and activity measurement should be adapted to plant species, and to the organs of the same plant, because of extreme weaknesses and instabilities of the enzyme. Different extraction and reaction media have been compared in order to define the best conditions for cotton callus nitrate reductase activity measurement. The use of potassium phosphate buffer, at pH 7.5 allowed for a better stabilization of the enzyme during extraction. The addition of 15 mM of glutamine in the extraction buffer stimulates significantly, in vitro, the reduction of nitrate. Enzyme activity is moreover optimal when 1 M of exogenous nitrate, as substrate, is added to the reaction medium. At these optimum conditions of nitrate reductase activity determination, the substrate was completely reduced after 20 min of enzyme incubation, and the greatest velocity of nitrate transformation into nitrite (ìg of NO2 -/min/g F.W) is observed when incubation period of enzyme is short (1 to 5 min).