Inhibition of nitrate reductase from spinach (Spinacea oleracea L.) leaf by adenosine nucleotides

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NADH-dependent nitrate reductase of spinach showed ping-pong kinetics. Inhibition by ADP in the absence of thiols increased during a transient phase during which kinetics changed from weak (K_i 20 mM), non-competitive and linear to comparatively strong (K_i 0.09 mM at 10 mM ADP), mixed with respect to NADH and non-linear (n_H = 1.7). Thiol compounds transformed the inhibition to competitive (K_i 0.18 mM at 10 mM ADP) and non-linear (n_H=2.3). Effective concentrations increased in order: GSH>mercaptoethanol=thioglycollate>cysteine, and were increased by dissolved oxygen or disulphide. Non-competitive inhibition by ribose-5-phosphate (K_i 3 mM) was abolished by glutathione. Cyclic 3',5'-AMP inhibited non-linearly (n_H=2.4) and comparably to or more than ADP (K_i 0.22 mM at 2.5 mM) but predominantly competitively except at low NADH concentrations. Cyclic 2',3'-AMP showed similar but weaker inhibition (K_i 1.4 mM) and more nearly linear (n_H=1.3). 5'-AMP inhibited competitively (K_i 1.2 mM at 10 mM) (n_H= 1.3). 5'-ATP showed weak mixed inhibition (K_i 5 mM at 10 mM) (n_H=1.3). Activity with BV° was scarcely inhibited by ADP, but phosphate activation with BV° or NADH were both inhibited by ADP. Physiological significance of inhibition by ADP is discussed and might lie between 3% in light and 25% in dark in special circumstances of thiol supply.
日本植物生理学会の論文