Inactivation of nitrate reductase involves NR-protein phosphorylation and subsequent 'binding' of an inhibitor protein

The function of two proteins (P67 and P100) required for the MgATP-dependent inactivation of nitrate reductase (NR) from spinach leaves (Spinacia oleracea L.) was studied. When NR was incubated with γ-[32P]ATP and P67, NR-protein was phosphorylated, but without a change in NR activity. Protein P100 by itself was neither able to phosphorylate nor to inactivate NR, and when added together with P67 it did not change the extent of NR phosphorylation. However, when NR was first phosphorylated with MgATP and P67, subsequent addition of P100 after removal of unreacted ATP caused an immediate NR inactivation. In presence of both P67 and P100 the time-course of ATP-dependent NR phosphorylation paralleled the time course of inactivation. The extent of NR phosphorylation and of NR inactivation (in the presence of P67 plus P100) was similarly affected by metabolites or high salt concentrations. Magnesium (Mg2+) played a dual role in the inactivation process: the phosphorylation of NR by P67 was strictly Mg2+-dependent. Further, phospho-NR (+ P100) was inactive only in the presence of Mg2+, but active in the presence of excess EDTA. Dephospho-NR appeared to be Mg2+-insensitive. The observations suggest that phosphorylation of NR by P67 is obligatory, but not sufficient for inactivation. In addition to protein phosphorylation, inactivation requires "binding" of an inhibitor protein (P100) to phospho-NR.