Adenine nucleotides are apparently involved in the light-dark modulation of spinach-leaf nitrate reductase

Nitrate reductase (NR; EC 1.6.6.1) in spinach (Spinacia oleracea L. cv. Polka F1) leaves showed reversible modulation, being activated in the light and inactivated in the dark (t/2 = 20–30 min). The large changes in enzyme activity during light-dark transients were observed only when assayed in buffers containing free Mg²⁺. In the presence of EDTA (5 mM), the enzyme activity was high and the light modulation was barely evident.The inactivation of NR in the dark could be totally prevented by anaerobiosis, or by feeding mannose or 2,4-dinitrophenol through the leaf petiole. All these treatments drastically decreased ATP levels and increased AMP levels in leaf extracts, thus pointing to a close correlation between adenine-nucleotide levels and NR activity. Treatment of leaves in the dark with 2,4-dinitrophenol or with anaerobiosis brought about an accumulation of nitrite, thus confirming that under these conditions NR remained active also in vivo. The in-vivo dark-inactivated enzyme was reactivated in vitro by preincubating a leaf extract with AMP in the presence of the myokinase inhibitor p¹,p⁵-di(adenosine 5)pentaphosphate. It is suggested that NR responds to artificially induced drastic changes in cytosolic adeninenucleotide levels, being active when ATP is low and AMP is high. Adenine nucleotides also appear to participate in the light-dark modulation of NR, but additional regulatory factors have to be postulated.