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Chapter 5,706

Inhibition of nitrate reductase from spinach spinacia oleracea leaf by adenosine nucleotides

Eaglesham, A.R.J.; Hewitt, E.J.

Plant and Cell Physiology 16(6): 1137-1149

1975


ISSN/ISBN: 0032-0781
Accession: 005705565

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 (Ki 20 mM), non-competitive and linear to comparatively strong (Ki 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 (Ki 0.18 mM at 10 mM ADP) and non-linear (nH = 2.3). Effective concentrations increased in order: glutathione > mercaptoethanol = thioglycollate > cysteine, and were increased by dissolved O2 or disulphide. Non-competitive inhibition by ribose-5-phosphate (Ki 3 mM) was abolished by glutathione. Cyclic 3',5'-AMP inhibited non-linearly (nH = 2.4) and comparably to or more than ADP (Ki 0.22 mM at 2.5 mM) but predominantly competitively except at low NADH concentrations. Cyclic 2',3'-AMP showed similar but weaker inhibition (Ki 1.4 mM) and more nearly linear (nH = 1.3). 5'-AMP inhibited competitively (Ki 1.2 mM at 10 mM) (nH = 1.3). 5'-ATP showed weak mixed inhibition (Ki 5 mM at 10 mM) (nH = 1.3). Activity with BV.degree. [reduced benzyl viologen] was scarcely inhibited by ADP, but phosphate activation with BV.degree. or NADH were both inhibited by ADP. The physiological significance of inhibition by ADP is discussed; it may range between 3% in light and 25% in dark in special circumstances of thiol supply.

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