Some Unusual Characteristics of Nitrate Reduction in Erythrina Senegalensis Dc.

NADPH nitrate reductase activity in higher plants has been attributed to the presence of NAD(P)H bispecific nitrate reductases and to the presence of phosphatases capable of hydrolyzing NADPH to NADH. To determine which of these conditions exist in barley (Hordeum vulgare L. cv. Steptoe), we characterized the NADH and NADPH nitrate reductase activities in crude and affinity-chromatography-purified enzyme preparations. The pH optima were 7.5 for NADH and 6 to 6.5 for the NADPH nitrate reductase activites. The ratio of NADPH to NADH nitrate reductase activities was much greater in crude extracts than it was in a purified enzyme preparation. However, this difference was eliminated when the NADPH assays were conducted in the presence of lactate dehydrogenase and pyruvate to eliminate NADH competitively. In most higher plant tissues, the reduction of nitrate to nitrite is attributed to NADH-specific NR2 (EC 1.6.6.1) (1, 2). However, NADPH NR activities have been reported in several species. NAD(P)H bispecific NR (EC 1.6.6.2) has been reported in maize (4), rice (13), soybeans (1,3,5,8,11), and Erythrina senegalensis (14). Wells and Hageman (17) 10). Zinc acetate and phenazine methosulfate were used to remove excess NAD(P)H from the assay medium to avoid interference with nitrite determination (12). It was necessary to agitate vigorously this mixture to ensure complete oxidation of pyridine nucleotides when NAD(P)H concentrations were greater than 200 JiM in the assay medium. Apparent Km values were determined by Lineweaver-Burk plots, except the Km for NADH, which was determined by the method of Halwachs (7).The NR assay buffer was modified in several studies. In the pH study, the assay buffer was 25 mm Tris, 25 mM maleic acid, and 25 mm phosphate. The pH was adjusted to the indicated value with KOH. In studies to determine the effects ofphosphate and fluoride upon NAD(P)H NR activities and to determine whether Pi increased during the NADPH assay of the purified NR, 50 mm Hepes was used at pH 7.5, and 50 mm maleic acid was used at pH 6.5. Pi was determined by the procedure of Taussky and Shorr (15). NR assays were also conducted in the presence of LDH and pyruvate to eliminate NADH competitively from the assay medium.NR Isolation. NR was purified by blue dextran-agarose affinity chromatography, as described by Kuo et al. (9). NADPH Phosphatase. Crude NR extracts were passed through Sephadex G-25 to remove nitrate and pyridine nucleotides. About 25 ml of G-25 were placed in a 30-ml colunm (2-cm diameter) and equilibrated with 25 mm Tris (pH 8.4), 1 mm EDTA, 10 JIM Na2MoO4, 5 JIM FAD, and 3 mm DTT. Crude extract (5 ml) was placed on the column and eluted with the equilibration buffer. A 4.5-ml fraction containing most of the protein was collected and used to estimate the rate of NADPH conversion to NADH. One ml of the G-25 fraction was incubated in 50 mm maleic acid (pH 6.5)

Section: Discussionmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

See 1 more Smart Citation

Pyridine Nucleotide Specificity of Barley Nitrate Reductase

Dailey¹,

Kuo²,

Warner³

1982

“…For samples from the PGO incubation medium, nitrate was assayed with nitrate reductase supplied by Sigma. In this procedure, nitrate is converted to nitrite which was determined using a combination of the procedures of Ida and Morita (7) and Stewart and Orebamjo (22). The assay mixture contained in a final volume of 1 mL, 0.4 mL of 0.8 mm fresh-made methylviologen in 250 mm potassium phosphate buffer (pH 7.0), 0.1 unit of nitrate reductase in 0.1 mL of 100 mM potassium phosphate (pH 7.0), 0. influence of pH can be accounted for by an increase in deprotonated guanidinium groups and a reaction with PGO through nucleophilic addition to the uncharged guanidinium residues (10,27,30).…”

Section: Measurement Of Nitrate Uptakementioning

confidence: 99%

Essential Arginine Residues in the Nitrate Uptake System from Corn Seedling Roots

Beevers

1990

Three dicarbonyl reagents were used to demonstrate the presence of an essential arginine residue in the N03-uptake system from corn seedling roots (Zea mays L., Golden Cross Bantam). Incubation of corn seedlings with 2,3-butanedione (0.125-1.0 millimolar) and 1,2-cyclohexanedione (0.5-4.0 millimolar) in the presence of borate or with phenylglyoxal (0.25-2.0 millimolar) at pH 7.0 and 300C resulted in a time-dependent loss of N03-uptake following pseudo-first-order kinetics. Second-order rate constants obtained from slopes of linear plots of pseudo-first-order rate constants versus reagent concentrations were 1.67 x 10-2, 0.68 x 10-2, and 1.00 x 10-2 millimolar per minute for 2,3-butanedione, 1,2-cyclohexanedione, and phenylglyoxal, respectively, indicating the faster rate of inactivation with 2,3-butanedione at equimolar concentration. Double log plots of pseudofirst-order rate constants versus reagent concentrations yielded slope values of 1.031 (2,3-butanedione), 1.004 (1,2-cyclohexanedione), and 1.067 (phenylglyoxal), respectively, suggesting the modification of a single arginine residue. The effectiveness of the dicarbonyl reagents appeared to increase with increasing medium pH from 5.5 to 8.0. Unaltered Km and decreased Vmax in the presence of reagents indicate the inactivation of the modified carriers with unaltered properties. The results thus obtained indicate that the N03-transport system possesses at least one essential arginine residue.

“…The pH optima for both the NADH and the NADPH nitrate reductase activities from nrla were approximately pH 7.7, which is slightly greater than the pH 7.5 optimum for the NADH activity and considerably greater than the pH 6.0 to 6.5 optimum for the NADPH activity of the wild-type enzyme. (3, 4,7,12,23,27,28). The NR from the wild-type barley is a NADH-specific enzyme (6).…”

mentioning

confidence: 99%

Characteristics of a Nitrate Reductase in a Barley Mutant Deficient in NADH Nitrate Reductase

Dailey¹,

Warner²,

Somers³

et al. 1982

A barley (Hordeum vulgare L.) mutant, narla (formerly AzM2), deficient in NADH nitrate reductase activity is, nevertheless, capable of growth with nitrate as the sole nitrogen source. In an attempt to identify the mechanism(s) of nitrate reduction in the mutant, nitrate reductase from narla was characterized to determine whether the residual activity is due to a leaky mutation or to the presence of a second nitrate reductase. The results obtained indicate that the nitrate reductase in narla differs from the wild-type enzyme in several important aspects. The pH optima for both the NADH and the NADPH nitrate reductase activities from nrla were approximately pH 7.7, which is slightly greater than the pH 7.5 optimum for the NADH activity and considerably greater than the pH 6.0 to 6.5 optimum for the NADPH activity of the wild-type enzyme. (3, 4,7,12,23,27,28). The NR from the wild-type barley is a NADH-specific enzyme (6).The objective of this study was to characterize the NR from the NR-deficient mutant, narla, to determine whether the enzyme is the same or different from the wild-type NR. MATERIALS AND METHODSPlant Material. Barley (Hordeum vulgare L.) seedlings were grown for 6 to 7 d in a growth chamber at 16°C under continuous light (300 ,uE m 2s-'). The NR-deficient mutants, narla (formerly Az12) and nar2a (formerly Az34), were induced and selected from the cv. Steptoe, as described by Warner et aL (32). Both mutants are homozygous and genetically stable (14).NR Extraction and Assay. Seedlings were extracted with a buffer (3 ml/g fresh weight leaf tissue) containing 25 mm Tris (pH 8.4), 1.5 mm EDTA, 4.0 mm DDT, and 5 ,LM FAD. The crude homogenates were centrifuged at 27,000g for 15 min, and the supernatant was saved (crude extract). NAD(P)H NR assays were conducted as described previously (30), except that 0.2 ml of a 1:1 mixture of 0.3 mm phenazine methosulfate and 1.0 M zinc acetate was used to stop the reaction (25). To insure complete oxidation of the NAD(P)H, this mixture was vigorously mixed particularly when the NAD(P)H concentrations in the assay were greater than 0.2 mm.Assays for reduced methylviologen NR activity were performed in a medium containing 25 mm K-phosphate (pH 7.5), 10 mM potassium nitrate, 0.2 mm methylviologen, and 3.2 mm sodium dithionite. Assays were conducted by adding the enzyme to the assay medium (2 ml final volume), incubating at 30°C for 1 min, initiating the reaction with 0.1 ml of the dithionite solution (14 mg/ml of 25 mm phosphate [pH 8.2]), and incubating at 30°C for 30 min. The assay was stopped by vigorously agitating the assay mixture until the methylviologen was oxidized (about 10 s). To reduce interference from substances derived from sodium dithionite, 0.2 ml of 1.5% (v/v)