Specificity for Nicotinamide Adenine Dinucleotide by Nitrate Reductase from Leaves

Abstract

Preliminary work revealed that nitrate reductase in crude extracts prepared from leaves of certain corn genotypes as well as soybeans could utilize NADPH as well as NADH as the electron donor. Isoelectric focusing and diethylaminoethyl cellulose chromatography confirmed previous findings that NADH and NADPH activities could not be separated, which suggests the involvement of a single enzyme. Nitrate reduction with both cofactors varies with plant species, plant age, and assay conditions. The ability of the nitrate reductase from a given genotype to utilize NADPH was associated with the amount of NADPHphosphatase in the extract. While diethylaminoethyl cellulose chromatography of plant extracts separated nitrate reductase from the bulk (90 %) of the phosphatase and caused a decrease in the NADPH activity, the residual level of phosphatase was sufficient to account for the apparent NADPH nitrate reductase activity. Addition of KH2PO4 and KF, inhibitors of NADPH-phosphatase activity in in vitro assays, caused a drastic reduction or abolishment of NADPH-mediated nitrate reductase activity but were without effect on NADH nitrate reductase activity. It is concluded that NADPH-nitrate reduction, in soybean and certain corn genotypes, is an artifact resulting from the conversion of NADPH to NADH by a phosphatase and that the enzyme in leaf tissue is NADH-dependent (E.C.1.6.6.1).While numerous investigations have shown that nitrate reductase extracted from a variety of higher plants has a specific or highly preferential requirement for NADH (2), the enzyme from soybean leaves apparently utilizes either NADH or NADPH with equal efficiency (1, 6). Investigations with the in vivo assay also suggest that the enzyme is NADH-dependent (12). Although we have reported that NR' from corn was NADH-specific (1, 9), recently we have observed that NR from leaf tissue of certain corn genotypes can also utilize NADPH as the electron donor. The level of NADPH activity observed in these initial experiments was a function of pH and assay buffer.It is possible that the NADPH-NR activity in both soybean (8), and Hevea brasiliensis (10).The objectives were: (a) to demonstrate the range of NADPH-dependent NR activity in relation to NADH-NR activity as affected by the type and pH of the assay buffer; (b) to determine if NR from corn genotypes that exhibited marked NADPH activity is a single enzyme as was shown for the soybean enzyme (1); and (c) to determine if the NADPH-NR activity in crude and partially purified preparations is an artifact arising from the presence of a phosphatase that hydrolyzes NADPH to NADH and P,.MATERIAL AND METHODS Plant Material and Culture. Leaf tissue from corn (Zea miiays L.), soybeans (Glycine max L. Merr. var. Hawkeye), pigweed (Amaranthus hybridus L.), and foxtail (Seteria faberii Herrm.) was harvested from young plants grown in growth chambers (16 hr light-2,500 ft-c at 32 C and 8 hr dark at 22 C) in vermiculite (Zonalite Co., Chicago, Ill.) subirrigated daily with Hoagland's No. 1 nutrient solution. ...

Identification of Hydroxypyruvate and Glyoxylate Reductases in Maize Leaves

“…Campbell [5]). The phosphatase-induced artefacts in measurements of leaf NADPH-nitrate reductase were first reported by Wells and Hageman (34).…”

Specificity for Nicotinamide Adenine Dinucleotide and Nicotinamide Adenine Dinucleotide Phosphate of Nitrate Reductase from the Salt-tolerant Alga Dunaliella parva

“…There was no preferential inhibition of the NADPH-dependent activity by either inorganic phosphate or fluoride as compared with the NADH-dependent activity. Such an inhibition could be expected if a phosphatase were associated with the ability to use NADPH (13). Furthermore nmol Pi were detected in the incubation mixture.…”

“…However, the ability to utilize NADPH as electron donor could be an artifact caused by the presence of a phosphatase in the crude extract which converted NADPH to NADH and Pi. as was recently shown for several higher plants (13). The following experiments were designed to determine whether this was the case for the nitrate reductase of D. parva as well.…”

“…10). The ability of the enzyme from several higher plants to use NADPH as well as NADH (2, 4) was recently shown ( 13) to be an artifact caused by the presence in the extract of a phosphatase-like activity which converted NADPH to NADH and Pi. In an earlier communication (8).…”

“…Ion exchange chromatography on DEAE-cellulose was carried out according to Wells and Hageman (13 Centre-Negev, Beer-Sheva, Israel x 40 cm) was equilibrated with 50 mm K-phosphate (pH 7.5). containing 1 mM L-cysteine and eluted with a linear gradient of 0 to 0.25 M K2S04 in the equilibrating buffer.…”