Yoshikiyo Oji

Nitrite reduction in barley-root plastids: Dependence on NADPH coupled with glucose-6-phosphate and 6-phosphogluconate dehydrogenases, and possible involvement of an electron carrier and a diaphorase

Plastids from roots of barley (Hordeum vulgare L.) seedlings were isolated by discontinuous Percoll-gradient centrifugation. Coinciding with the peak of nitrite reductase (NiR; EC 1.7.7.1, a marker enzyme for plastids) in the gradients was a peak of a glucose-6-phosphate (Glc6P) and NADP+-linked nitrite-reductase system. High activities of phosphohexose isomerase (EC 5.3.1.9) and phosphoglucomutase (EC 2.7.5.1) as well as glucose-6-phosphate dehydrogenase (Glc6PDH; EC 1.1.1.49) and 6-phosphogluconate dehydrogenase (6PGDH; EC 1.1.1.44) were also present in the isolated plastids. Thus, the plastids contained an overall electron-transport system from NADPH coupled with Glc6PDH and 6PGDH to nitrite, from which ammonium is formed stoichiometrically. However, NADPH alone did not serve as an electron donor for nitrite reduction, although NADPH with Glc6P added was effective. Benzyl and methyl viologens were enzymatically reduced by plastid extract in the presence of Glc6P+ NADP+. When the plastids were incubated with dithionite, nitrite reduction took place, and ammonium was formed stoichiometrically. The results indicate that both an electron carrier and a diaphorase having ferredoxin-NADP+ reductase activity are involved in the electron-transport system of root plastids from NADPH, coupled with Glc6PDH and 6PGDH, to nitrite.

Sensitive spectrophotometric method for the determination of ethylenediaminetetraacetic acid in foods

A sensitive spectrophotometric method for the determination of ethylenediaminetetraacetic acid (EDTA) in foods is described. The method involves the reaction of EDTA with Fe3+ to produce the EDTA-Fe chelate, followed by the removal of excess of Fe3+ by a chelate extraction technique using chloroform and N-benzoyl-N-phenylhydroxylamine and the formation of a chromophore with 4,7-diphenyl-1,10-phenanthroline-disulfonic acid. The calibration graph was linear in the range 0.5-40.0 micrograms cm-3 of EDTA with a slope of 21.1. The relative standard deviation at 10 micrograms cm-3 of EDTA was 1.6% (n = 10). There was no interference from most of the common ingredients of commercial foods. More than 90% of EDTA added at two levels was recovered from real samples. The method was applied to the determination of EDTA in various foods, and the results obtained were compared with those given by high-performance liquid chromatography.

Purification and characterization of NADH-nitrate reductase from leaves of 2-row barley, and its activity as affected by some metabolites

Summary NADH-nitrate reductase (EC 1.6.6.1) was purified 4260-fold from leaves of 2-row barley ( Hordeum vulgare L.). The purification procedure involved extraction with Tris-HCl buffer, ammonium sulfate fractionation, adsorption on Phenyl Sepharose CL-4B and hydroxyapatite, affinity chromatography on Blue Sepharose CL-6B and followed by gel filtration on TSK-Gel (G-4000SW) by using high-performance liquid chromatography. The purified preparation was nearly homogenous as revealed by disc gel electrophoresis. It had a specific activity of 37 μmol nitrite formed min −1 · mg protein −1 at 30°C; the highest specific activity among nitrate reductases so far isolated from a variety of barley cultivars. Its native molecular weight was estimated as 225,000. The 130,000 daltons subunit found on SDS gels seemed to be the actual nitrate reductase subunit, the smaller proteins seeming to be degradation products. The respective apparent Km values for NADH and nitrate at pH 7.5 (optimum for the activity) were 3.8 and 270 μM. FAD was required for the maximal activity of the enzyme; the apparent Km for FAD was 8 nM. Carbamyl phosphate behaved as a competitive inhibitor with respect to nitrate. Among adenine derivatives, only ADP inhibited the activity, the inhibition being competitive with NADH. Inhibition by pyridoxal-5′-phosphate was uncompetitive with nitrate. No inhibition was observed with pyridoxal itself, or with pyridoxine or pyridoxamine-5′-phosphate. Other metabolites tested were without effect on the activity. It seems that barley leaf nitrate reductase has a lysine residue at its active site. Heavy metals (Cu 2+ , Zn 2+ , Co 2+ , {ie1} and {ie2}) caused a strong inhibition at 1 mM. {ie3} (10 mM) activated the activity.

Nitrate reduction in root and shoot and exchange of reduced nitrogen between organs in two-row barley seedlings under light-dark cycles.

Nitrate reduction in roots and shoots and exchange of reduced N between organs were quantitatively estimated in intact 13-d-old seedlings of two-row barley (Hordeum vulgare L. cv. Daisengold) using the 15N-incorporation model (A. Gojon et al. (1986) Plant Physiol. 82, 254–260), except that NH+4was replaced by NO-2. N-depleted seedlings were exposed to media containing both nitrate (1.8 mM) and nitrite (0.2 mM) under a light-dark cycle of 12:12 h at 20°C; the media contained different amounts of 15N labeling. Experiments were started either immediately after the beginning (expt. 1) or immediately prior to the end (expt. 2) of the light period, and plants were sampled subsequently at each light-dark transition throughout 36 h. The plants effectively utilized 15NO-3and accumulated it as reduced 15N, predominantly in the shoots. Accumulation of reduced 15N in both experiments was nearly the same at the end of the experiment but the accumulation pattern in roots and shoots during each 12-h period differed greatly depending on time and the light conditions. In expt. 1, the roots accounted for 31% (light), 58% (dark), and 9% (light) of nitrate reduction by the whole plants, while in expt. 2 the contributions of the root were 82% (dark), 20% (light), and 29% (dark), during each of the three 12-h periods. Xylem transport of nitrate drastically decreased in the dark, but that of reduced N rather increased. The downward translocation of reduced 15N increased while nitrate reduction in the root decreased, whereas upward translocation decreased while nitrate reduction in the shoot increased. We conclude that the cycling of reduced N through the plant is important for N feeding of each organ, and that the transport system of reduced N by way of xylem and phloem, as well as nitrate reduction by root and shoot, can be modulated in response to the relative magnitude of reduced-N demands by the root and shoot, with the one or the other predominating under different circumstances.

Enzymic method for the determination of nitrite in meat and fish products

A simple spectrophotometric method for the determination of nitrite is described. Nitrite is measured enzymically through its reaction with nitrite reductase coupled with NADH. The entire enzymic procedure required 15 min to complete. The calibration curve was linear in the range 0.1-10 micrograms cm-3 nitrite with a slope of 6.25. The relative standard deviation at 5 microgram g-1 was 1.7% (n = 5). The method greatly simplifies the procedure of nitrite determination and enables the routine inspection of a number of samples with very little laboratory equipment. A comparison study showed that the method was superior to the GC method for samples containing large amounts of reducing substances while good agreement was achieved between both methods for other foods.

Enzymic method for the spectrophotometric determination of aspartame in beverages

A sensitive spectrophotometric method for the determination of aspartame in beverages is described. The method involves the enzymic conversion of aspartame into formaldehyde by the alpha-chymotrypsin-alcohol oxidase system, followed by the formation of a chromophore with 4-aminopent-3-en-2-one. The calibration graph was linear in the range 2.0-30.0 micrograms ml-1 of aspartame. Many common ingredients of beverages do not interfere with the proposed method. The method was applied to the determination of the aspartame content of various real samples, and the results obtained were compared with those given by high-performance liquid chromatography.

Role of xylem sap nitrate in the regulation of nitrate reductase gene expression in leaves of barley (Hordeum vulgare L.) seedlings

Abstract The role of xylem sap nitrate in the regulation of nitrate reductase (NR) in leaves was investigated in barley (Hordeum vulgare L.) seedlings. Upon the exposure of barley roots to nitrate at concentrations ranging from 0.1 to 5 mm, rapid accumulation of NADH-specific NR mRNA was observed in both leaves and roots. In the same treatment, the concentration of xylem sap nitrate also rapidly increased during the early feeding period and reached a steady state at concentration ranging from 15 to 50 mm. Patterns of NR mRNA accumulation in leaves were closely linked with the increase in the nitrate concentration in the xylem sap, but not with the amount of nitrate stored in the leaf tissues. High levels of NR mRNA in intact leaves were observed only when the xylem sap nitrate reached mm levels. Supply of different concentrations of nitrate to detached leaves also indicated that mm levels of nitrate supplied through the xylem were required for adequate accumulation of NR mRNA in leaves. When nitrate-grown seedlings were transferred to a nitrate-free medium, a parallel decline in both the concentration of xylem sap nitrate and NR mRNA level was observed, while the nitrate concentrations in leaf tissues remained constant. These results indicate that continuous supply of mm levels of nitrate through the xylem is important for maintaining the high levels of NR mRNA in barley leaves.

Appearance of endopeptidases during the senescence of cucumber leaves

Abstract In cucumber ( Cucumis sativus L.) leaves at different ontogenic stages, a differential appearance of three major endopeptidases was observed by employing activity staining using gelatin as a substrate. On the basis of this observation, we discussed their physiological roles in senescing leaves. The most active endopeptidase in young mature leaves was a glutamyl endopeptidase with a pI of 4.5. It might be involved in active protein catabolism in young leaves because its activity became maximal just after the leaf had fully expanded and when protein and ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) contents rapidly decreased. An endopeptidase with a pI of 4.3 was not observed in young leaves, however, it was highly active in senescing leaves. Interestingly, its activity in cotyledons was eliminated when the upper metabolically active leaves were removed. This implies that the appearance of this enzyme is regulated by the presence of sink tissues, and it is involved in the degradation of protein in senescing leaves facilitating N transfer to upper developing leaves. A trypsin-like endopeptidase with a pI of 5.0 showed relatively constant activity during the whole period. This endopeptidase has been shown to be inhibited by arginine, guanidino compounds and Mg 2+ , therefore, it might exist constitutively and its activity might be regulated mainly at a post-translational level responding to nutrient and environmental conditions.

Effect of nitrogen application at the flowering stage on the quality of soybean seeds

Abstract Application of nitrogen (N) fertilizer at the flowering stage changed the contents of storage compounds in seeds of Soybean (Glycine max L. cvs Enrei and Tamahomare). The effects of the N application on the maturation of soybean seeds were examined by comparing changes in the contents of amino acids, sugars, water, protein, and oil in seeds from N‐dressed plants (NDS) with those from undressed plants (UDS) during maturation. The application resulted in a decrease of contents of total and some amino acids (glutamine and asparagine) in developing seeds except for at the early maturation stage and in a decreased protein content of mature seeds. On the other hand, the N application led to faster accumulation of oil in developing seeds and to an increased oil content of mature seeds. Based on these results, it was concluded that the N application at the flowering stage changed the composition of solutes imported by developing seeds and resulted in variations in the contents of storage compounds. The r...

Determination of glycerol in foods by high-performance liquid chromatography with fluorescence detection

Abstract A high-performance liquid chromatographic method for the determination of glycerol in foods is described. The method involves the conversion of glycerol into formaldehyde by sequential enzymatic reactions (glycerokinase, glycerol-3-phosphate oxidase, catalase), followed by the derivatization of formaldehyde with 4-amino-3-penten-2-one. The calibration graph was linear in the range. 0.1-4.0 μg/ml of glycerol. Many common ingredients of foods did not interfere. More than 90% of glycerol added at three levels was recovered from several foods. The method is simple and accurate. The detection limit was 1.0 μg/g when 5 g of sample were assayed.