NADH-dependent Nitrate Reductase Research Articles

Induction of NADH-dependent nitrate reductase activity in cucumber cotyledons by de novo synthesis of the enzyme

Summary Under non-inducing conditions, nitrate reductase (EC 1.6.6.1, NADH: nitrate oxidoreductase) is not detectable by enzymatic and immunological methods in cotyledons of cucumber (Cucumis sativus L.). Nitrate reductase activity induced by nitrate or pR stress is due to de novo synthesis of nitrate reductase apoprotein, as was shown by inhibition assays using antibodies directed against nitrate reductase of tobacco (Nicotiana tabacum). During the fast induction by ammonium nitrate, enzymatically inactive nitrate reductase apoprotein is accumulated in the middle phase of the induction curve. There is no accumulation of inactive nitrate reductase during the retarded induction by citrate at pR 2.5.

Synthesis and Accumulation of Nitrite in Soybean Nodules Supplied with Nitrate

Nodulated soybean plants (Glycine max [L.] Merr) were grown in sand culture without combined N or with a continuous supply of nitrate in nutrient solution. Moderate nitrate concentration (30 milligrams N per liter) had little effect on nodule weight/plant while high nitrate concentration (100 milligrams N per liter) depressed nodule weight/plant by 70 to 80% with harvests 30 to 60 days after planting and initiation of nitrate treatments.The effect of nitrate supply on ammonium, amino, and ureide nitrogen concentrations in nodules was small and inconsistent. In contrast, nitrate and nitrite concentrations in nodules were directly proportional to nitrate supply and inversely proportional to nodule weight/plant. Correlations between nitrate or nitrite concentration in nodules and nodule weight/plant were highly significant.Cytosol from soybean nodules was found to contain NADH-dependent nitrate reductase activity (typical activity was 0.1 micromole per milligram protein x hour). A Rhizobium japonicum mutant (derived from strain 61A76) lacking nitrate reductase was employed to show that the cytosol enzyme activity is of host origin. Growth of nodules formed by the mutant lacking nitrate reductase was inhibited by nitrate. These nodules did contain nitrite although concentrations of nitrite (about 0.3 microgram N per gram fresh weight) were low relative to nitrite concentrations (about 1.5 microgram N per gram fresh weight) in nodules formed by R. japonicum strain 61A76. The overall results support the idea that the depression of legume nodule growth by nitrate is directly related to the metabolism of nitrate in nodules.

Histochemical localization of nitrate reductase.

NADH-dependent nitrate reductase (E.C. 1.6.6.1) was ultrastructurally localized in norflurazon-treated and control soybean cotyledons [Glycine max (L.) Merr.] by a method based upon the increase in osmiophilia due to the formation of an azo dye. The reaction product was observed in small vesicles throughout the cytoplasm. An apparent transport of nitrite to the plastid, the site of nitrite reduction, may occur through fusion of the nitrite-containing vesicles with the chloroplast envelope. Plants grown in tungstate lacked nitrate reductase activity as measured by standard assay procedures, and showed no increase in osmiophilia, suggesting a degree of specificity of this cytochemical procedure.

Sulphite- and NADH-dependent Nitrate Reductase from Thiobacillus denitrificans

SUMMARY: A membrane-bound nitrate reductase from Thiobacillus denitrificans can utilize either sulphite or NADH as an electron donor. The sulphite-dependent nitrate reductase activity was released from the membrane by treatment with sodium deoxycholate. Cytochrome c and FAD were separated from the solubilized enzyme by heat treatment and subsequent chromatography on DEAE-cellulose. The bacterial cytochrome c and a preparation of horse-heart cytochrome c served as electron mediators to the solubilized sulphite-dependent nitrate reductase activity with apparent K m values of 1·5 and 1·3 μ m respectively. The NADH-linked enzymic activity, which was unstable during storage, was re-activated with reduced glutathione. It was also inactivated after treatment with deoxycholate but this effect was reversed by menadione. A possible scheme for electron transport for the sulphite- and NADH-dependent enzyme is proposed.

Induction of nitrate reductase by succinic acid-2,2-dimethylhydrazide in cucumber cotyledons

Summary Succinic acid-2,2-dimethylhydrazide (B-Nine) induced nitrate reductase (NR) activity in detached, etiolated cucumber cotyledons as well in light as in darkness. Optimal concentration of B-Nine for NR induction was 25 mM. The compound inhibited growth and chlorophyll synthesis in the cotyledons. Chloramphenicol (CAP) and benzylaminopurine (BAP) also induced measurable NR activity in the cotyledons grown in darkness. B-Nine when applied at sub-optimal concentrations produced a synergistic effect with BAP and CAP: e.g. CAP (5 mM), B-Nine (5 mM) and CAP + B-Nine treated cotyledons produced about 4, 2 and 30 nmoles NO2− h−1 cotyledon−1, respectively. KNO3 did not potentiate effectivity of B-Nine or CAP as inducers of NR. Since cycloheximide reduced NR activity in the B-Nine and CAP treated cotyledons, it was inferred that the enzyme was synthesized de novo in cytoplasma. KNO3, BAP and B-Nine induced NADH-dependent NR (EC 1.6.6.1); B-Nine induced also some activity of NADPH-NR which was not detected in BAP + KNO3 induced cotyledons.