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 sudden darkness, and rapidly recovered upon reillumination. However, the amount of NR protein, estimated by western blots, did not fluctuate during short-term light/dark/light transitions. This suggests that rapid changes of NR activity in response to light/dark regimes are due to reversible modulation of the protein and not to de novo synthesis/degradation. In mannose-fed leaves, such light/dark changes in NR activity were 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 inactivation of NR. The radiolabel of γ-³²P] ATP was incorporated into NR protein in vitro and the labelling of NR was blocked by K-252a. On the other hand, extractable NR from darkened leaves was activated by incubation at 30°C without further additions. The in vitro activation of NR was prevented by calyculin A, a potent and specific inhibitor of protein phosphatase. Moreover calyculin A abolished the in vivo activation of NR by illumination. Our results confirm a regulatory system by phosphorylation/dephosphorylation of NR. The data also suggest that the activity of NR depends on the relative phosphorylation/dephosphorylation activities subtly controlled in response to photon flux density.