Nariaki Wakiuchi

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.

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.

Molecular and crystal structure of galactinol dihydrate [1-O-(α-d-galactopyranosyl)-myo-inositol dihydrate]

The crystal structure of galactinol dihydrate has been determined by X-ray diffraction. The crystal belongs to the orthorhombic system, space group P2(1)2(1)2, a = 15.898(6), b = 19.357(5), c = 5.104(4) A, and Z = 4. The structure was refined to R = 0.044 for 1818 observed structure amplitudes. The primary hydroxyl group exhibits twofold orientational disorder. The linkage conformation is close to those of alpha-(1 --> 4) linkages in methyl alpha-maltotrioside tetrahydrate and erlose trihydrate. Although there is no interring hydrogen bond in galactinol, an indirect interring hydrogen bond including a water molecule is present. The observed conformation is additionally stabilized by the indirect interring hydrogen bond. The global minimum in the relaxed-residue energy map based on the MM3(92) force-field is close to the observed conformation in the crystal structure. All hydroxyl, ring and water oxygen atoms are involved in a complex three-dimensional hydrogen-bonding network.

Properties of the Vanadate-Sensitive ATPase of the Plasma Membrane Vesicles from Rice Roots

Abstract The plasma membrane vesicles were purified by aqueous two phase partitioning from rice roots (Oryza sativa L. cv. Nipponbare). The ATPase activity of the purified plasma membrane fraction (U3 fraction) increased more than 4-fold by the addition of Triton X-100, and was not inhibited by trypsin digestion pretreatment. Vanadate inhibited 70% of the ATPase activity of the Triton-washed U3 fraction. The K m value was 1.45 mM, and vanadate and ADP inhibited the activity uncompetitively and competitively, respectively. The ATPase activity was dependent on Mg2+, and Co2+ could be completely substituted for Mg2+, while Fe2+ and Mn2+ partially. KCl increased the activity by 20% with saturation at 5 mM. The order of the stimulation by monovalent cations was NH4 +>K+>Li+, choline+>Na+. The optimum pH for the activity was 6. The vesicles of the U3 fraction were reconstituted with deoxycholate and soybean phospholipids. The proton pumping activity of the fraction was inhibited by vanadate. Based on the result...

Fate of minor free amino acids and phospholipids in crude tallow during steam splitting

Minor free amino acids and phospholipids contained in crude tallow were monitored during steam splitting of crude tallow. The bulk of the phospholipids was found in the glycerol sidestream after splitting. Phosphatidic acid and phosphatidylcholine were present in both crude tallow and the glycerol fraction. Phosphatidylserine and phosphatidylethanolamine present in crude tallow were hydrolized with the glycerides. Because of this hydrolysis, high amounts of serine and ethanolamine are found in the fatty acid and glycerol fractions. In addition to constituent amino acids of proteins present in crude tallow, other biological amino acids such as taurine and ornithine were also present.

Nitrite reduction system in barley roots: existence of an electron carrier operating in an electron-transport sequence from glucose-6-phosphate to nitrite reductase

An electron carrier that can be substitutes for ferredoxin in a cytochrome c reduction system has been identified in extracts prepared from the roots of barley (Hordeum vulgare L. cv. Daisen-gold) seedlings. The root electron carrier (REC) had a red color and was able to transport electrons from a NADPH-regenerating system (glucose-6-phosphate, NADP, and glucose-6-phosphate dehydrogenase) to cytochrome c in a reaction catalyzed by ferredoxin-NADP reductase (EC 1.6.7.1) from spinach leaves. It had absorption maxima at 412 and 467 nm, and the absorption peak at 412 nm shifted to 419 nm upon reduction with dithionite. REC could transport electrons from dithionite to nitrite reductase (EC 1.7.7.1) purified from the same roots, and also operated as an electron transporter in the presence of spinach leaf ferredoxin-NADP reductase from glucose-6-phosphate to the nitrite reductase system, which is considered to be the in vivo nitrite-reduction system in the roots. REC could not be distinguished from spinach leaf ...

Characterization of proton pumping and ATPase activities in microsomal fractions from barley roots

Abstract The properties of proton pumping and ATPase activities of barley (Hordeum vulgare L. cv. Daisen-gold) roots were investigated using microsomal preparations and partially purified ATPase. Quenching of acridine orange fluorescence was used to measure the proton pumping activity of microsomal vesicles collected on Dextran T-70. Addition of ATP·Mg3+ caused the quenching of the fluorescence. The quenching returned to the original level by addition of protonophores ((NH4)2SO4, gramicidin). The proton pumping activity was dependent on KCl, and was inhibited by KNO3. The ATPase enzyme was solubilized from microsomal preparations with 0.2% Zwittergent 3–14, and partially purified by DEAE-cellulose column chromatography. The ATPase activity was dependent on Mg2+, and inhibited by vanadate. The activity of the purified ATPase which was derived from the plasma membranes in the microsomal preparations, was insensitive to K+, and stimulated by Ca2+ in the absence of Mg2+. These results suggest that the proton ...

Reconstitution and Characterization of Plasma Membrane H^+-ATPase Activity from Rice Roots

Abstract Plasma membrane vesicles were purified from rice (Oryza sativa L. cv. Nippon-bare) roots using aqueous two-phase partitioning. The purified vesicles were solubilized with deoxycholate and reconstituted with soybean phospholipids by gel filtration. lonophores stimulated the ATPase activity of the reconstituted vesicles. Inhibition of the ATPase activity by vanadate increased from 77 to 93% by the reconstitution. These results indicate that the reconstituted vesicles were sealed and the vanadate-sensitive ATPase activity was purified during the reconstitution. The ATPase activity of the reconstituted vesicles was characterized. Only ATP was effectively hydrolyzed. Apparent Km value was 1.26 mM. Divalent cations were necessary for the activity, and their stimulation was in the following order: Mg2+>Fe2+>Mn2+, Co2+≫Ca2+. Half-maximal inhibition by vanadate occurred at 5 μM. Monovalent ions stimulated the activity, and cations and anions were effective in the order of NH4 + >K+, Rb+>Na+> Li+> choline+...

Changes in DNA level of growing barley seedlings.

Abstract The DNA content of barley (Hordeum vulgare L. cv. Daisen-gold) seedlings was determined by fluorometry with a fluorescent dye, 4′, 6-diamidino-2-phenyl- indole (DAPI) based on the method developed by Baer et al. (Plant Physiol., 70, 999-1003, 1982). Extraction and determination of DNA were investigated for application to barley seedlings. NaCl (2.0 M) and Sarkosyl (1%) were required in the homogenization buffer to obtain a higher DNA yield. The rate of increase of the content of DNA in growing barley seedlings was lower than that of fresh and dry weight. The distribution of DNA in a barley plant was analyzed. Higher DNA contents were found in the second leaf and root tip region. The relationship between the DNA content and cell volume was discussed.