PHOSPHORYLATION DEPHOSPHORYLATION OF KOMATSUNA (BRASSICA-CAMPESTRIS) LEAF NITRATE REDUCTASE IN-VIVO AND IN-VITRO IN RESPONSE TO ENVIRONMENTAL LIGHT CONDITIONS - EFFECTS OF PROTEIN-KINASE AND PROTEIN PHOSPHATASE INHIBITORS

Activity of nitrate reductase (NR; EC 1.6.6.1) in leaves of Komatsuna (Brassica campestris L. ssp. rapifera cv. Osome) was decreased by sudd en darkness, and rapidly recovered upon reillumination. However, the a mount of NR protein, estimated by western blots, did not fluctuate dur ing short-term light/dark/light transitions. This suggests that rapid changes of NR activity in response to light/dark regimes are due to re versible modulation of the protein and not to de novo synthesis/degrad ation. In mannose-fed leaves? such light/dark changes in NR activity w ere not observed. When extracts from illuminated leaves were incubated with MgATP, NR activity decreased in a time-dependent manner. K-252a, a specific inhibitor of protein kinases, prevented the in vitro inact ivation of NR. The radiolabel of [gamma-P-32] ATp was incorporated int o NR protein in vitro and the labelling of NR was blocked by K-252a. O n the other hand: extractable NR from darkened leaves was activated by incubation at 30 degrees C without further additions. The in vitro ac tivation of NR was prevented by caiyculin A, a potent and specific inh ibitor of protein phosphatase. Moreover calyculin A abolished the in v ivo activation of NR by illumination. Our results confirm a regulatory system by phosphorylation/dephosphorylation of NR. The data also sugg est that the activity of NR depends on the relative phosphorylation/de phosphorylation activities subtly controlled in response to photon flu x density.