Immunochemical characterization and localization of nitrate reductase in norflurazon‐treated soybean cotyledons

Immunochemical procedures were used to characterize and localize NADH:nitrate reductase (NR; EC 1.6.6.1) in cotyledons of norflurazon‐treated soybeans [ Glycine max (L.) Merr. cv. ‘Hill’]. Antiserum prepared to NR isolated from Chlorella strongly reacted against NR from norflurazon‐treated cotyledons. This serum inhibited the NR activity in crude extracts of norflurazon‐treated soybean cotyledons by 98% even at a 1:2000 dilution of crude serum. Pre‐immune serum had no effect on the activity. These data indicate that there are similar antigenic determinants at the active site of both Chlorella and norflurazon‐treated soybean NR. Whole cotyledons were homogenized in lithium dodecyl sulfate‐containing buffer, electrophoretically separated and blotted to nitrocellulose. When the blots were reacted with the anti‐NR serum only a single protein (M r = 98 kdalton) was visualized. Immunofluorescence studies on fixed tissue sections revealed intense fluorescence in the cytoplasm. Weaker reactions were associated with organelles tentatively identified as plastids. Pre‐immune serum controls were completely unstained using immunocytochemical procedures.