Jiro Sekiya

Purification and Properties of Soluble and Bound γ-Glutamyltransferases from Radish Cotyledon

Soluble and cell wall bound γ-glutamyltransferases (GGTs) were purified from radish (Raphanus sativus L.) cotyledons. Soluble GGTs (GGT I and II) had the same M r of 63,000, and were composed of a heavy subunit (M r, 42,000) and a light one (M r, 21,000). The properties of GGT I and II were similar. Bound GGTs (GGT A and B) were purified to homogeneity from the pellet after the extraction of soluble GGTs. GGT A and B were monomeric proteins with an M r of 61,000. The properties of GGT A and B were similar. Thus, bound GGTs were distinguished from soluble GGTs. The optimal pHs of soluble and bound GGTs were about 7.5. Both soluble and bound GGTs utilized glutathione, γ-L-glutamyl-p-nitroanilide, oxidized glutathione and the conjugate of glutathione with monobromobimane as substrates, and were inhibited by acivicin, but soluble GGTs were also distinguished from bound GGTs with regard to these properties.

Cytokinin-induced shoot formation

Abstract Zeatin, (±)-dihydrozeatin and optically active cytokinins (asymmetric carbon α to the exocyclic nitrogen) were tested for their ability to induce development of shoots in tobacco callus. Zeatin and dihydrozeatin were equally active. The levorotatory compounds tested were active in inducing shoot formation but the corresponding dextrorotatory compounds were inactive at all concentrations tested. These findings suggest that the group attached to the N 6 position of cytokinins binds to a receptor site to bring about organ formation.

Subcellular Localization and Possible Functions of γ-Glutamyltransferase in the Radish (Raphanus sativus L.) Plant

Previously we reported the purification of soluble γ-glutamyltransferases (GGTs) from radish cotyledon. Subcellular fractionation of radish cells revealed that soluble GGT is a vacuolar enzyme. Acivicin, a GGT inhibitor, mediated the in vivo catabolism inhibition of the glutathione S-conjugate generated from endogenous glutathione and exogenously supplied monochlorobimane. Thus soluble GGT is possibly involved in the catabolism of glutathione S-conjugates.

Synthesis of Hydroxymethylglutathione from Glutathione and L-Serine Catalyzed by Carboxypeptidase Y

Hydroxymethylglutathione (γ-L-glutamyl-L-cysteinyl-L-serine; hmGSH) occurs in many species belonging to the family Gramineae, but the biosynthetic pathway for hmGSH has not been identified. We found that carboxypeptidase Y (CPY), but not carboxypeptidase A, catalyzed hmGSH synthesis from glutathione and L-serine in vitro at acidic pH. CPY also catalyzed methylglutathione synthesis from glutathione and L-alanine. These findings suggested that a carboxypeptidase-like enzyme may be involved in hmGSH synthesis in vivo.

Nitrogen fixation of Sesbania rostrata: Contribution of stem nodules to nitrogen acquisition

Abstract Nitrogen contents, nodule numbers, and nodule dry weights of 6-week-oId Sesbania rostrata plants grown in sand culture with only root nodules, only stem nodules or with both were compared and the root nodules were found to contribute to nitrogen acquisition more significantly than the stem nodules. Similar findings were obtained in 15N2-fixing experiments. An 8-week-old plant with both stem and root nodules fixed 1.50 mg nitrogen in a 12 h light period, while the fixation decreased to 1.15 mg nitrogen after the removal of the stem nodules, suggesting that root nodules played major role in nitrogen fixation. However, acetylene-reducing activities per nodule dry weight were higher in the stem nodules. Under flooding conditions, the aerenchyma tissues contributed to about 40% of N2 transport to root nodules, and 60% was supplied through stem.

Rapid purification and characterization of cystine lyase b from broccoli inflorescence

Abstract We found three isoforms (a, b, and c) of cystine lyase in broccoli ( Brassica oleracea var. italica ) inflorescence tissues. Cystine lyase b, the most abundant isoform, was rapidly purified to homogeneity. The native enzyme had a M r of 160,000 and composed of four identical subunits with a M r of 40,000. Thiocysteine and pyruvate were confirmed as reaction products. The purified cystine lyase b utilized l -cystine and S -alkyl l -cysteine sulfoxide as substrates. Other properties of cystine lyase b were almost similar to those of the isoform a as reported previously. Cystine lyase a and b were localized in cytosolic and/or vacuole fraction. A high activity of cystine lyase was detected in some Allium species in addition to Cruciferae plants.

Purification and Characterization of Cystine Lyase a from Broccoli Inflorescence

One of the three isoforms of an enzyme degrading L-cystine was purified to homogeneity from broccoli (Brassica oleracea var. italica) inflorescences, with use of a sensitive assay based on derivatization of a reaction product with monobromobimane. The reaction product with a thiol group was found to be thiocysteine from results of liquid chromatography-mass spectrometry and high-resolution mass spectrometry. Pyruvate was also a reaction product, formed in equimolar amounts. The purified enzyme catalyzed β-elimination of L-cystine to yield thiocysteine, pyruvate and possibly ammonia, so it was cystine lyase a. L-Cystine but not D-cystine was a substrate of the enzyme. S-Methyl L-cysteine sulfoxide and S-ethyl L-cysteine sulfoxide were substrates but were less suitable than L-cystine. L- and D-cysteine and also cystathionine were not substrates. The purified enzyme (Mr 186,000) was composed of four identical subunits (Mr 45,000) and was pyridoxal 5-phosphate-dependent.

Tissue specificity of mitochondrial F0F1-ATPase activity of Lilium longiflorum plant.

A large difference was found in the activities of oligomycin‐sensitive mitochondrial F0F1‐ATPase isolated from different tissues of Lilium longiflorum plants. The enzyme activity of F0F1‐ATPase in pollen was the highest, while that in bulbs was the lowest. When ATPases were cross‐reconstituted from F1‐ATPases and F1‐depleted submitochondrial particles (SMP), ATPases reconstituted from F1‐depleted pollen SMP showed the higher activity regardless of the source of F1‐ATPase. Fatty acid compositions of phospholipids in SMP were also different between bulbs and pollen. These suggest that the F0 portion and/or its environment are important for regulation of F0F1‐ATPase activity in L. longiflorum plant.

Purification and Characterization of Dipeptidase Hydrolyzing L -Cysteinylglycine from Radish Cotyledon

Dipeptidase activity was detected in the soluble fraction of radish (Raphanus sativus L.) cotyledon, and the purified enzyme had a specific activity of 7.32 nkat/mg protein for hydrolyzing L-cysteinylglycine. The dipeptidase was found to be a hexameric metalloenzyme, composed of homological 55 kDa-subunits. This is the first glutathione catabolism-related dipeptidase isolated from higher plants.

Growth characteristics of Sesbania species under adverse edaphic conditions in relation to use as green manure in Japan

Abstract Edaphic-stress tolerance of three Sesbania species, Sesbania rostrata, S. cannabina, and S. speciosa was evaluated in terms of growth. Plants exhibited the most vigorous growth in August, the hottest days in Japan, while in July and September, the growth decreased by 30 to 80%; S. speciosa showed the strictest requirement for temperature. When the medium pH was 3.5, S. cannabina was the most tolerant to low pH. At pH 8, growth was reduced to 60% for all the species. S. speciosa appeared to be less sensitive to Al (1 mol m-3). Copper (1.6 and 6.4 mmol m-3) damaged plants irrespective of species. S. speciosa was less sensitive and S. cannabina slightly sensitive to drought. S. rostrata yielded 16.8 Mg ha-1 dry matter in 13 weeks under field conditions, with 426 kg nitrogen ha-1 in the aerial parts. Timing of seed sowing was the determinant for a higher biomass production.