In Vitro Stability of Nitrate Reductase from Wheat Leaves

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Abstract
A nitrate reductase (EC 1.6.6.1)-inactivating factor was isolated from 8-day-old wheat leaves. The purification schedule involved ammonium sulfate precipitation, Sephadex G-100 filtration, DEAE-cellulose chromatography and Sephadex G-150 filtration. No accurate assessment could be made as to the degree of purification relative to crude extract as the inactivating factor could not be detected in crude extract. However a 2446-fold purification was achieved from the ammonium sulfate fraction to the pooled enzyme from the Sephadex G-150 step. The inactivating factor was heat-labile and had a MW of 37,500. The inactivating factor was particularly sensitive to the divalent metal chelators, 1,10-phenanthroline and bathophenanthroline. Evidence indicated that Fe2+ may be the functional metal. The trypsin inhibitors N-.alpha.-p-tosyl-L-lysine chloromethyl ketone and .alpha.-N-benzoyl-L-arginine were inhibitory. However, phenylmethyl sulfonyl fluoride, an inhibitor of serine peptide hydrolases, was not inhibitory. Neither casein nor hemoglobin nor a range of artificial substrates were hydrolyzed by the inactivating factor. Highly purified wheat leaf nitrite reductase (EC 1.7.99.3) and ribulose 1,5-bisphosphate carboxylase:oxygenase (EC 4.1.1.39) were not affected by the nitrate reductase-inactivating factor. The inactivating factor was more active toward the NADH-nitrate reductase compared to either of the component enzymic activities flavin adenine mononucleotide-nitrate reductase and methyl viologen-nitrate reductase. The NADH-ferricyanide reductase (diaphorase) component was the least sensitive.