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

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Abstract
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 (K1 20 mM), non-competitive and linear to comparatively strong (K1 0.09 mM at 10 mM ADP), mixed with respect to NADH and non-linear (nH = 1.7). Thiol compounds transformed the inhibition to competitive (K1 0.18 mM at 10 mM ADP) and non-linear (nH = 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 (K1 3 nua) was abolished by glutathione. Cyclic 3′,5′-AMP inhibited non-linearly (nH=2.4) and comparably to or more than ADP (K1 0.22 mM at 2.5 mM) but predominantly competitively except at low NADH concentrations. Cyclic 2′,3′-AMP showed similar but weaker inhibition (K1 1.4 mM) and more nearly linear (nH =1.3). 5′-AMP inhibited competitively (K1 1.2 mM at 10 mM) (nH = 1.3). 5′-ATP showed weak mixed inhibition (K1 5 nun at 10 mM) (nH = l.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.

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