RAPID MODULATION OF NITRATE REDUCTASE IN LEAVES AND ROOTS - INDIRECT EVIDENCE FOR THE INVOLVEMENT OF PROTEIN PHOSPHORYLATION DEPHOSPHORYLATION/

Nitrate reductase activity (NRA; NADH-nitrate reductase, E.C. 1.6.6.1) has been measured in extracts from leaves of spinach (Spinacia olerac ea L.) in response to rapid changes in illumination, or supply of CO2 or oxygen. Measured in buffers containing magnesium, NRA from leaves d ecreased in the dark and increased again upon illumination. It decreas ed also, when CO2 was removed in continuous light, and was reactivated when CO2 was added. Nitrate reductase (NR) from roots of pea (Pisum s ativum L.) was also rapidly modulated in vivo. It increased under anae robiosis and decreased in air or pure oxygen. The half time for inacti vation or reactivation in roots and leaves was 5 to 30 min. When spina ch leaves were harvested during a normal day/night cycle, extractable NRA was low during the night, and high during daytime. However, at any point of the diurnal cycle, NR could be brought to a similar maximum activity by preincubation of the desalted leaf extract with AMP and/or EDTA. Thus, the observed diurnal changes appeared to be mainly a cons equence of enzyme modulation, not of protein turnover. In vivo, the re activation of the inactivated enzyme from both leaves and roots was pr evented by okadaic acid, an inhibitor of certain protein phosphatases. Artificial lowering of the ATP-levels in leaf or root tissues by anae robiosis (dark), mannose or the uncoupler carbonyl cyanide m-chlorophe nyl hydrazon (CCCP), always brought about full activation of NR. By pr eincubating crude leaf or root extracts with MgATP, NR was inactivated in vitro. Partial purification from spinach leaves of two enzymes wit h molecular masses in the 67 kD and 100 kD range, respectively, is rep orted. Both participate in the ATP-dependent inactivation of NR. Allto gether these data indicate that NR can be rapidly modulated by reversi ble protein phosphorylation/dephosphorylation, both in Shoots and in r oots.