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981.
λ-Glutamylcysteine synthetase in higher plants: catalytic properties and subcellular localization 总被引:1,自引:0,他引:1
λ-Glutamylcysteine synthetase activity (EC 6.3.2.2) was analysed in Sephacryl S-200 eluents of extracts from cell suspension
cultures ofNicotiana tabacum L. cv. Samsun by determination of λ-glutamylcysteine as its monobromobimane derivative. The enzyme has a relative molecular
mass (Mr) of 60000 and exhibits maximal activity at pH 8 (50% at pH 7.0 and pH9.0) and an absolute requirement for Mg2+. With 0.2mM Cd2+ or Zn2+, enzyme activity was reduced by 35% and 19%, respectively. Treatment with 5 mM dithioerythritol led to a heavy loss of activity
and to dissociation into subunits (Mr 34000). Buthionine sulfoximine andl-methionine-sulfoximine, known as potent inhibitors of λ-glutamylcysteine synthetase from mammalian cells, were found to be
effective inhibitors of the plant enzyme too. The apparent Km values forl-glutamate,l-cysteine, and α-aminobutyrate were, respectively, 10.4mM, 0.19 mM, and 6.36 mM. The enzyme was completely inhibited by glutathione
(Ki=0.42 mM). The data indicate that the rate of glutathione synthesis in vivo may be influenced substantially by the concentration
of cysteine and glutamate and may be further regulated by feedback inhibition of λ-glutamylcysteine synthetase by glutathione
itself. λ-Glutamylcysteine synthetase is, like glutathione synthetase, localized in chloroplasts as well as in the cytoplasm.
Chloroplasts fromPisum sativum L. isolated on a Percoll gradient contained about 72% of the λ-glutamylcysteine synthetase activity in leaf cells and 48%
of the total glutathione synthetase activity. In chloroplasts ofSpinacia oleracea L. about 61% of the total λ-glutamylcysteine synthetase activity of the cells were found and 58% of the total glutathione
synthetase activity. These results indicate that glutathione synthesis can take place in at least two compartments of the
plant cell.
Dedicated to Professor A. Prison on the occasion of his 80th birthday 相似文献
982.
The loading of amino acids and nitrate into the xylem was investigated by collection and analysis of root-pressure exudate from the cut hypocotyl stumps of seedlings of Ricinus communis L. Glutamine was found to be the dominant amino acid in the exudate and also to be the amino acid which is transferred to the xylem most rapidly and accumulated to the greatest extent. The comparison between uptake and xylem loading showed significant differences in specificity between these two transport reactions, indicating a different set of transport systems. Nitrate is transferred to the xylem at a higher relative rate than any amino acid despite the great nitrate-storage capacity of the root system. Thus the supply of nitrate to Ricinus plants leads to enhanced nitrogen allocation to the shoots. 相似文献
983.
Nodules of cowpea (Vigna unguiculata (L.) Walp. cv. Vita 3:Bradyrhizobium CB 756) from 28-d-old plants cultured for 23 d with their root systems maintained in O2 levels from 1 to 80% (v/v, in N2) in the external gas phase showed a range of structural changes which have been interpreted in relation to an over- or under-supply
of O2. A response to the partial pressure of O2 in the gas phase (pO2) was noted with respect to nodule size, lenticel development, the relative distributions of cortical and infected central
tissue, the differentiation of cortex, especially the inner cortex, the frequency and size of infected and uninfected interstitial
cells, the volume of extracellular spaces both in cortex and infected tissue, and in the frequency of bacteroids. As a consequence
of these changes the surface area of inner cortex relative to the nitrogenase-containing units of fixing tissue (infected
cells or bacteroids) was increased by as much as 20-fold. Effectiveness of bacteroid functioning increased from 0.10 ± 0.02
· 10-9 μmol acetylene reduced per bacteroid in air-grown nodules to 0.9 ± 0.16 · 10-9 (same units) per bacteroid in those cultured in 1% O2.
This work was supported by a grant from the Australian Research Council (to C.A.A.) and an Australian International Development
Assistance Bureau postgraduate fellowship (to F.D.D.). The authors wish to thank Dr. W.F.C. Blumer for his considerable help
with morphometric analysis, Dr. J. Kuo for guidance in the use of histological techniques, and to Dr. J.S. Pate for the suggestion
that lenticel development might be quantified by surface staining of nodules. 相似文献
984.
Phosphoenolpyruvate carboxylase (PEPCase; EC 4.1.1.31) activity was found to be modulated by light and darkness when measured in the presence of K+, which had been added to induce swelling of guard-cell protoplasts (GCPs) from Vicia faba L., whereas no modulation was detected in the absence of K+ (PEPcase activity remained constant at 1.5±0.15 pmol PEP metabolized · GCP–1 ·h–1; subsequently, pmol GCP–1 ·h–1 will be used). The activity of PEPCase increased by 100% (from 1.5 to 3 pmol·protoplast–1·h–1) in darkness and by 200% (from 1.7 to 5 pmol·protoplast–1· h–1) in light and oscillations in activity of these magnitudes were repeated at intervals of 2 min (dark) and 2.5 min (light) for a period of 10 min during K+-induced increase in the volume of GCPs. The oscillations were reflected in changes in malate-pool sizes determined in plastids, mitochondria and the supernatant fraction (consisting of the cytosol and the vacuole). Malate probably functioned as a mitochondrial substrate, thus supplying ATP for K+ uptake and the swelling of the protoplasts. On the basis of the present paper and previous results (H. Schnabl and B. Michalke 1988, Life Sci. Adv. Plant Physiol. 7, 203–207) involving adenine nucleotidepool sizes in fractionated GCPs, a model is proposed to explain the cause-effect relationship between K+, PEPCase, the cytosolic and mitochondrial malate levels and ATP levels during the K+-induced increase of GCP volume.Abbreviations GCP
dtguard-cell protoplast
- PEP
phosphoenol-pyruvate
- PEPCase
PEP carboxylase
The authors thank Professor Hermann Schnabl, University of Stuttgart (FRG), for his assistance in applying the graph theory analysis. This work was supported by Deutsche Forschungsgemeinschaft to H.S. 相似文献
985.
986.
Cell-suspension cultures of two chickpea (Cicer arietinum L.) cultivars, resistant (ILC 3279) and susceptible (ILC 1929) to the fungus Ascochyta rabiei (Pass.) Lab., showed differential accumulation of the phytoalexins medicarpin and maackiain, and transient induction of related enzyme activities after application of an A. rabiei-derived elicitor. The chalcone-synthase (CHS) activity (EC 2.3.1.74) which is involved in the first part of phytoalexin biosynthesis exhibited a maximum 8–12 h after elicitation in the cells of both cultivars. Concomitant with the fivefold-higher phytoalexin accumulation, CHS activity increased twofold in the cells of the resistant cultivar. The maximum of the elicitor-induced CHS-mRNA activity was determined 4 h after onset of induction in the cultures of both cultivars, although in cells of cultivar ILC 3279 this mRNA activity was induced at a level twofold higher than that in cells of the susceptible race ILC 1929. Investigations of CHS isoenzymes by two-dimensional gel electrophoresis of immunoprecipitated in-vitro-translated protein indicated the presence of five proteins. In the cells of both cultivars only two of the isoenzymes were induced after elicitor treatment. Analysis of the total in-vitro-translated proteins by two-dimensional gel electrophoresis showed that the constitutively expressed patterns of mRNA activities in the cell cultures of the two cultivars were identical. After elicitation, considerably more translatable mRNAs were induced in the cells of cultivar ILC 3279. The few induced proteins, and their respective mRNA activities, which could be detected in the cells of the susceptible cultivar, all existed in the cells of the resistant cultivar, too. One highly induced protein (Mr 18 kDa) found in the cells of cultivar ILC 3279 reached its maximum mRNA activity 6 h after elicitor application. The amount of this protein was hardly increased in the cells of the susceptible cultivar. This protein appears to be excreted from the cells into the growth medium.Abbreviations CHS
chalcone synthase
- IEF
isoelectric focussing
- ILC
international legume chickpea
- PR-protein
pathogenesis-related protein
- SDS-PAGE
sodium dodecyl sulfate-polyacrylamide gel electrophoresis
Financial support by Deutsche Forschungsgemeinschaft and Fonds der Chemischen Industrie is gratefully acknowledged. The authors thank Dr. K. Hahlbrock (Max-Planck-Institut für Züchtungsforschung, Köln, FRG) for provision of antisera and the International Centre for Agricultural Research in the Dry Areas (Aleppo, Syria) for plant material. 相似文献
987.
Spatiotemporal patterns of expression of the cell-surface arabinogalactan-protein epitope defined by monoclonal antibody JIM4 (J.P. Knox et al., 1989, Development 106, 47–56) have been characterized by indirect immunofluorescence during the process of somatic embryogenesis in Daucus carota L. The JIM 4 epitope (J4e) occurred on cells established in culture from hypocotyl explants which appeared to derive, at least in part, from the epidermal cells of the hypocotyl. Cultures maintained in the presence of 2,4-dichlorophenoxyacetic acid developed proembryogenic masses of which only infrequent cells at the surface expressed J4e. Sub-culture at a low cell density and withdrawl of the synthetic auxin resulted in an increase in J4e expression in most surface cells and most abundantly in surface layers of cells at the future shoot end of developing embryos. The transition to heart-shaped embryos occurred concurrently with the expression of J4e by groups of cells beneath the developing cotyledons, at the junction of the future root and shoot. At this stage, J4e was also expressed by a single well-defined layer of cells at the surface of the embryos. Advancement to the mature torpedo stage was accompanied by the expression of the epitope on cells forming two regions of the future stele and of cells associated with the cotyledonary provascular tissue characteristic of the carrot seedling. At this stage there was substantially less expression of the marker antigen by epidermal cells, although infrequent expression by isolated cells of the epidermis was maintained. The correlation of J4e expression with the development and distinction of plant tissue patterns during somatic embryogenesis indicates a role for plasma-membrane arabinogalactan proteins in these processes.Abbreviations AGP
arabinogalactan protein
- 2,4-D
2,4-di-chlorophenoxyacetic acid
- J4e
JIM 4 epitope
- PEM
proembryogenic mass
We thank Andrew Davis for photographic assistance and Roger Pennell for useful discussions. 相似文献
988.
B. P. Forster R. P. Ellis 《TAG. Theoretical and applied genetics. Theoretische und angewandte Genetik》1990,80(6):806-809
Summary Aneuploid stocks, which included Triticum aestivum/alien, disomic, chromosome addition lines, wheat/alien, ditelosomic, chromosome addition lines, and the available aneuploids of Chinese Spring wheat, were used to locate genes that influence milling energy requirement (ME). Genes that affected ME were found on all seven homoeologous chromosome groups. The addition of complete wheat chromosomes 1B, 1D, 2A, 2D, 5B, 6B, 7B and 7D increased ME. Positive effects were also found in specific chromosome arms: 1BS, 2DS, 5AS, 5BS and 6BL. Wheat chromosome 3B conditioned low ME and the gene(s) responsible was located on the short arm. Other negative effects were attributed to wheat chromosome arms 4BL, 4DL, 5DS and 6DS. Alien chromosome additions that conferred high ME included 2H, 5H, 6H and 7H of barley, Hordeum vulgare and 2R, 2R, 4R, 4RL, 6R, 6RL and 7RL of rye, Secale cereale. Those that conferred a low ME included 1H
ch of H. chilense, and 6u and 7u of Aegilops umbellulata, 5R and 5RS of S. cereale and 5R
m and 5R
mS of S. montanum. Although the control of ME is polygenic, there is a major effect of genes located on the short arms of homoeologous group 5 chromosomes. 相似文献
989.
J. A. C. VERKLEIJ P. KOEVOETS J. VAN'T RIET R. BANK Y. NIJDAM W. H. O. ERNST 《Plant, cell & environment》1990,13(9):913-921
Abstract. The effect of cadmium on growth of Cd-tolerant and -sensitive plants of Silene vulgaris and on the production of metal-binding compounds in both types of plants was studied. The Cd-content of the roots and the Cd-root/shoot ratio was higher in Cd-tolerant plants. A Cd-binding compound (Cd-BC) with an apparent molecular mass of 14.5 kD was isolated from the roots of Cd-tolerant and -sensitive plants, grown in 40 mmol m−3 Cd for 21 d. More than 60% of the total Cd in the roots was associated with this compound. Determination of the amino-acid content of the purified Cd-containing compound from both types of plants showed that they possessed a similar amino-acid composition to that of phytochelatins. Only the bis- and tris-forms were present. The amount of Cd and sulphide associated with phytochelatin was greater in tolerant plants than in sensitive ones suggesting that an increased sulphide content of complexes containing peptide, sulphide and Cd may form the basis of evolved Cd-tolerance in Silence vulgaris. 相似文献
990.
S. Abramov Y. Aharonowitz M. Harnik R. Lamed A. Freeman 《Enzyme and microbial technology》1990,12(12):982-988
The development of a continuous anaerobic process for stereospecific Δ4-3-keto-steroid reduction by immobilized Clostridium paraputrificum cells cells is described. Following a study on conditions for cell growth and sporulation, spores of C. paraputrificum were aseptically immobilized in PAAH beads. Conditions for cell growth and induction in the immobilized state were determined, as well as the medium composition required to maintain a stabilized immobilized cell population. The effect of the concentration of ethylene glycol added as selected cosolvent on reaction kinetics, substrate solubility, specific activity, and cell growth, was investigated. A 10% (v/v) cosolvent input provided maximal activity along with enhanced solubility of the steroidal substrate. It was shown that cell growth was enhanced in the presence of the added cosolvent in addition to its effect on substrate solubility and enzymic activity. The immobilized cells readily performed Δ4, as well as 3-keto steroid reduction of several steroids, including ADD, AD, 16-dehydroprogesterone, progesterone, and hydrocortisone. It was shown that repeated batch-wise reduction cycle—in the presence of the cosolvent—resulted in rapid loss of activity, while the continuous uninterrupted process permitted the attaining of full bioconversion level, maintained stable for at least the period of 5 days of continuous operation tested. 相似文献