Determination of Nitrate Reductase Activity in Barley Leaves and Roots

The inactivation of nitrate reductase in the leaves and roots of barley (Hordeum vulgare L. cv. Mazurka) during and after extracting was investigated. At 0 °C in the absence of casein, 25 per cent of 'total', i.e. maximal in vitro , nitrate reductase activity was lost during the 2 min extraction process, followed by a slower loss of activity while the extract was stored in ice. Activity was maintained by adding a minimum of 1 per cent casein to the extraction medium containing 01 m phosphate (pH 7-5), 1 mM EDTA and 1 mM dithiothreitol. Nitrate reductase was stable for several hours in these extracts, but declined in a first order manner in the absence of dithiothreitol. Casein also prevented the initial loss while making root extracts, but had less effect during storage. Using casein and thiols, nitrate reductase activity in light, (as product of maximal in vitro rates and wt g-1) in leaves was 98 per cent of the total activity in 31 -day-old plants grown with full nutrient in water culture and 60-day-old field-grown plants receiving no fertilizer. Field-grown plants, however, exhibited only 17 per cent of the activity of culture-grown plants. Nitrate reductase in leaves of barley plants grown in water culture had a diurnal rhythm. During the first 3 h of the light period, activity increased to 1 *3 x the 'dark' value. This was followed by a temporary decrease and then by another increase to a maximum of 1-7 x the 'dark' value, occurring about 8 h after illumination. Activity then decreased during the rest of the light period and in darkness.