Antibodies to assess phosphorylation of spinach leaf nitrate reductase on serine 543 and its binding to 14‐3‐3 proteins

To monitor site-specific phosphorylation of spinach leaf nitrate reductase (NR) and binding of the enzyme to 14-3-3 proteins, serum antibodies were raised that select for either serine 543 phospho- or dephospho-NR. The dephospho-specific antibodies blocked NR phosphorylation on serine 543. The phospho-specific antibodies prevented NR binding to 14-3-3s, NR inhibition by 14-3-3s, NR dephosphorylation on serine 543, and did not precipitate 14-3-3s together with NR. Together, this confirms that 14-3-3s bind to NR at hinge 1 after it has been phosphorylated on serine 543. The amounts of individual NR forms were determined in leaf extracts by immunoblotting and immunoprecipitation. The phosphorylation state of NR on serine 543 increased 2-3-fold in leaves upon a light/ dark transition. Before the transition, one-third of NR was already phosphorylated on serine 543 but was not bound to 14-3-3s. Phosphorylation of serine 543 seems not to be enough to bind to 14-3-3s in leaves.