INACTIVATION OF NITRATE REDUCTASE INVOLVES NR-PROTEIN PHOSPHORYLATIONAND SUBSEQUENT BINDING OF AN INHIBITOR PROTEIN

The function of two proteins (P67 and P100) required for the MgATP-dep endent inactivation of nitrate reductase (NR) from spinach leaves (Spi nacia oleracea L.) was studied. When NR was incubated with gamma-[P-32 ]ATP and P67, NR-protein was phosphorylated, but without a change in N R activity. Protein P100 by itself was neither able to phosphorylate n or to inactivate NR, and when added together with P67 it did not chang e the extent of NR phosphorylation. However, when NR was first phospho rylated with MgATP and P67, subsequent addition of P100 after removal of unreacted ATP caused an immediate NR inactivation. In presence of b oth P67 and P100 the time-course of ATP-dependent NR phosphorylation p aralleled the time course of inactivation. The extent of NR phosphoryl ation and of NR inactivation (in the presence of P67 plus P100) was si milarly affected by metabolites or high salt concentrations. Magnesium (Mg2+) played a dual role in the inactivation process: the phosphoryl ation of NR by P67 was strictly Mg2+-dependent, Further, phospho-NR (P100) was inactive only in the presence of Mg2+, but active in the pre sence of excess EDTA. Dephospho-NR appeared to be Mg2+-insensitive. Th e observations suggest that phosphorylation of NR by P67 is obligatory , but not sufficient for inactivation. In addition to protein phosphor ylation, inactivation requires ''binding'' of an inhibitor protein (P1 00) to phospho-NR.