A thioredoxin-activated fructose-1,6-bisphosphatase from the cyanobacterium Synechococcus 6301

Fructose-1,6-bisphosphatase was isolated from the cyanobacterium Synechococcus 6301 by acid precipitation, ammonium-sulfate fractionation, and Sephadex gel chromatography. The purified enzyme needed thiols and MgCl₂ for activity. The following K_m-values were obtained: a) for fructose-1,6-bisphosphate: 1.7 mM; b) for MgCl₂: 12.5 mM; c) for dithiocrythritol: 0,56 mM; d) for glutathione: 14 mM; e) for mercaptoethanol: 22 mM; f) for cysteine: 50 mM. Thioredoxin B isolated from this organism will activate this fructose-1,6-bisphosphatase. The K_m of thioredoxin B for this fructose-1,6-bisphosphatase was determined to be 1.7 M, endicotiy that thioredoxin might activate the fructose-1,6-bisphosphatase in Synechococcus in vivo.     

Specific inhibition of tetrapyrrole biosynthesis by 3-amino 2,3-dihydrobenzoic acid (gabaculin) in the cyanobacteriumSynechococcus 6301

Gabaculin (3-amino 2,3-dihydrobenzoic acid) inhibited the growth of cyanobacteria but not of other prokaryotes. Exposure of growing cultures ofSynechococcus 6301 to 50 M gabaculin resulted in an immediate and complete inhibition of the synthesis of chlorophylla and phycocyanin. With 8 M gabaculin, tetrapyrrole synthesis was suppressed for approximately 10 h and then resumed at a lower rate than in untreated organisms. The effect of 50 M gabaculin was reversed by transferring organisms to inhibitor-free medium; chlorophylla synthesis began within 5 h and exponential growth was re-established after about 25 h. Compared with 4,6-dioxoheptanoic acid (DA) and laevulinic acid (LA), gabaculin was a much more potent inhibitor of tetrapyrrole synthesis inSynechococcus 6301. The catalytic activity of -aminolaevulinic acid (ALA) dehydratase in vitro was inhibited by DA and LA but not by 1 mM gabaculin. However, the specific activity of the dehydratase was much lower in organisms exposed to the inhibitor for 36 h. Growing cultures and cell suspensions ofSynechococcus 6301 exposed to DA excreted appreciable quantities of ALA. In contrast, relatively small amounts of ALA accumulated in the presence of gabaculin alone and this inhibitor blocked the excretion of ALA caused by DA. This suggests that the primary effect of gabaculin is the specific inhibition of the C₅ pathway for the biosynthesis of ALA.Abbreviations ALA -aminolaevulinic acid - DA 4,6-dioxoheptanoic acid - LA laevulinic acid - GABA -aminobutyric acid     

Contrasting modes of photosynthetic enzyme regulation in oxygenic and anoxygenic prokaryotes

Enzymes that are regulated by the ferredoxin/thioredoxin system in chloroplasts — fructose-1,6-bisphosphatase (FBPase), sedoheptulose-1,7-bisphosphatase purified from two different types of photosynthetic prokaryotes (cyanobacteria, purple sulfur bacteria) and tested for a response to thioredoxins. Each of the enzymes from the cyanobacterium Nostoc muscorum, an oxygenic organism known to contain the ferredoxin/thioredoxin system, was activated by thioredoxins that had been reduced either chemically by dithiothreitol or photochemically by reduced ferredoxin and ferredoxin-thioredoxin reductase. Like their chloroplast counterparts, N. muscorum FBPase and SBPase were activated preferentially by reduced thioredoxin f. SBPase was also partially activated by thioredoxin m. PRK, which was present in two regulatory forms in N. muscorum, was activated similarly by thioredoxins f and m. Despite sharing the capacity for regulation by thioredoxins, the cyanobacterial FBPase and SBPase target enzymes differed antigenically from their chloroplast counterparts. The corresponding enzymes from Chromatium vinosum, an anoxygenic photosynthetic purple bacterium found recently to contain the NADP/thioredoxin sytem, differed from both those of cyanobacteria and chloroplasts in showing no response to reduced thioredoxin. Instead, C. vinosum FBPase, SBPase, and PRK activities were regulated by a metabolite effector, 5-AMP. The evidence is in accord with the conclusion that thioredoxins function in regulating the reductive pentose phosphate cycle in oxygenic prokaryotes (cyanobacteria) that contain the ferredoxin/thioredoxin system, but not in anoxygenic prokaryotes (photosynthetic purple bacteria) that contain the NADP/thioredoxin system. In organisms of the latter type, enzyme effectors seem to play a dominant role in regulating photosynthetic carbon dioxide assimilation.     

Acetate uptake by the unicellular cyanobacteria Synechococcus and Aphanocapsa

Acetate uptake by strains of Synechococcus and Aphanocapsa in short experiments required light, and was strongly inhibited by m-dichlorocarbonyl cyanide phenylhydrazone and dichlorophenyl dimethyl urea. Acetate carbon was distributed in amino acids and in the acyl portion of lipids in the same way as during growth experiments when CO₂ was available, but the reduced incorporation in the absence of CO₂ was primarily into the lipid fraction. An apparent K _m for uptake by Synechococcus and for Aphanocapsa 6308 of 20 and 180 M at pH 7.4 was obtained; corresponding V _max values were 6 and 11 nmol x min^-1 x mg protein^-1. Uptake with Synechococcus was affected by pH, with affinity decreased and maximal rate increase with rising pH. Acetate uptake was not affected by propionate or butyrate when both were added at the same time, but a light and concentration dependent inhibition developed if suspensions were preincubated with propionate. Acetate carbon moved rapidly into acid insoluble material, but after 10–15 s 75% or more of the recovered intracellular counts were in acetyl CoA. Counts in this compound were reduced by preincubation with propionate.Kinetic measurements of acetyl CoA synthetase in fractionated cell extracts gave values for K _m of about 50 M for acetate, 5 mM for propionate, 100 M for CoA and 0.38 mM for ATP. The internal pool of free CoA was measured to be about 20 M, and was reduced by preincubation with propionate. This suggests that the activity of CoA-mediated reactions may be regulated by the availability of this cofactor.Abbreviations Used CCCP m-Dichlorocarbonyl cyanide phenyl hydrazone - DCMU dichlorophenyl dimethyl urea - TCA trichloroacetic acid - Tris trishydroxymethyl amino methane - HEPES N-2-hydroxyethylpiperazine-N-2-ethane-sulfonic acid     

The influence of temperature on glucose-6-phosphate dehydrogenase and 6-phosphogluconate dehydrogenase and the regulation of these enzymes in a mesophilic and a thermophilic cyanobacterium

The activities and kinetics of the enzymes G6PDH (glucose-6-phosphate dehydrogenase) and 6PGDH (6-phosphogluconate dehydrogenase) from the mesophilic cyanobacterium Synechococcus 6307 and the thermophilic cyanobacterium Synechococcus 6716 are studied in relation to temperature. In Synechococcus 6307 the apparent K _m's are for G6PDH: 80M (substrate) and 20M (NADP⁺); for 6PGDH: 90M (substrate) and 25M (NADP⁺). In Synechococcus 6716 the apparent K _m's are for G6PDH: 550M (substrate) and 30M (NADP⁺); for 6PGDH: 40M (substrate) and 10M (NADP⁺). None of the K _m's is influenced by the growth temperature and only the K _m's of G6PDH for G6P are influenced by the assay temperature in both organisms. The idea that, in general, thermophilic enzymes possess a lower affinity for their substrates and co-enzymes than mesophilic enzymes is challenged.Although ATP, ribulose-1,5-bisphosphate, NADPH and pH can all influence the activities of G6PDH and 6PGDH to a certain extent (without any difference between the mesophilic and the thermophilic strain), they cannot be responsible for the total deactivation of the enzyme activities observed in the light, thus blocking the pentose phosphate pathway.Abbreviations G6PDH glucose-6-phosphate, dehydrogenase - 6PGDH 6-phosphogluconate dehydrogenase - G6P glucose-6-phosphate - 6PG 6-phosphogluconate - RUDP ribulose-1,5-bisphosphate - Tricine N-Tris (hydroxymethyl)-methylglycine     

The ferredoxin-thioredoxin system of a green alga,Chlamydomonas reinhardtii

The components of the ferredoxin-thioredoxin (FT) system of Chlamydomonas reinhardtii have been purified and characterized. The system resembled that of higher plants in consisting of a ferredoxin-thioredoxin reductase (FTR) and two types of thioredoxin, a single f and two m species, m1 and m2. The Chlamydomonas m and f thioredoxins were antigenically similar to their higher-plant counterparts, but not to one another. The m thioredoxins were recognized by antibodies to both higher-plant m and bacterial thioredoxins, whereas the thioredoxin f was not. Chlamydomonas thioredoxin f reacted, although weakly, with the antibody to spinach thioredoxin f. The algal thioredoxin f differed from thioredoxins studied previously in behaving as a basic protein on ion-exchange columns. Purification revealed that the algal thioredoxins had molecular masses (M_rs) typical of thioredoxins from other sources, m1 and m2 being 10700 and f 11 500. Chlamydomonas FTR had two dissimilar subunits, a feature common to all FTRs studied thus far. One, the 13-kDa (similar) subunit, resembled its counterpart from other sources in both size and antigenicity. The other, 10-kDa (variable) sub-unit was not recognized by antibodies to any FTR tested. When combined with spinach, (Spinacia oleracea L.) thylakoid membranes, the components of the FT system functioned in the light activation of the standard target enzymes from chloroplasts, corn (Zea mays L.) NADP-malate dehydrogenase (EC 1.1.1.82) and spinach fructose 1,6-bisphosphatase (EC 3.1.3.11) as well as the chloroplast-type fructose 1,6-bisphosphatase from Chlamydomonas. Activity was greatest if ferredoxin and other components of the FT system were from Chlamydomonas. The capacity of the Chlamydomonas FT system to activate autologous FBPase indicates that light regulates the photosynthetic carbon metabolism of green algae as in other oxygenic photosynthetic organisms.Abbreviations DEAE diethylaminoethyl - ELISA enzyme-linked immunosorption assay - FBPase fructose 1,6-bisphosphatase - Fd ferredoxin - FPLC fast protein liquid chromatography - FTR ferredoxin-thioredoxin reductase - FT system ferredoxin-thioredoxin system - kDa kilodaltons - M_r relative molecular mass - NADP-MDH NADP-malate dehydrogenase - SDS-PAGE sodium dodecyl sulfate-polyacrylamide gel electrophoresis This work was supported in part by a grant from the National Aeronautics and Space Administration. We would like to thank Don Carlson and Jacqueline Girard for their assistance with cell cultures.     

Effect of thidiazuron on vegetative tissue-derived somatic embryogenesis and flowering of bamboo Bambusa edulis

Current research on somatic embryogenesis of bamboo uses reproductive tissue as explants. However, it was hard to obtain the explant. Shoots of a local accession (3–4 m high) were used for multiple shoot production. In order to obtain embryogenic callus, nodal and internodal tissues from in vitro plantlets were placed on Murashige and Skoog (MS) medium supplemented with 9.2 M kinetin (KN), 13.6 M 2,4-dichlorophenoxyacetic acid (2,4-D), 0.1% (v/v) coconut milk, and 6% (w/v) sucrose. We studied the effects of sucrose and thidiazuron (TDZ) on callus proliferation. Optimal additives to the MS medium for embryogenic callus proliferation were 0.046 M TDZ, 13.6 M 2,4-D and 3% (w/v) sucrose. TDZ also promoted the germination of bamboo somatic embryos. The germination rate of the somatic embryos exceeded 80% on MS-based medium supplemented with 0.455M TDZ. Naphthaleneacetic acid (NAA) reduced germination. Well-developed plantlets were successfully transferred to soil. There was no albino mutant in subsequent culture. In vitro regenerants and potted plants flowered, but no seeds were produced.     

Liver metabolism in cold hypoxia: a comparison of energy metabolism and glycolysis in cold-sensitive and cold-resistant mammals

The effects of cold hypoxia were examined during a time-course at 2 °C on levels of glycolytic metabolites: glycogen, glucose, glucose-1-phosphate, glucose-6-phosphate, fructose-6-phosphate, fructose-1,6-bisphosphate, phosphoenolpyruvate, pyruvate, lactate and energetics (ATP, ADP, AMP) of livers from rats and columbian ground squirrels. Responses of adenylate pools reflected the energy imbalance created during cold hypoxia in both rat and ground squirrel liver within minutes of organ isolation. In rat, ATP levels and energy charge values for freshly isolated livers were 2.54 mol·g^-1 and 0.70, respectively. Within 5 min of cold hypoxia, ATP levels had dropped well below control values and by 8 h storage, ATP, AMP, and energy charge values were 0.21 mol·g^-1, 2.01 mol·g^-1, and 0.17, respectively. In columbian ground squirrels the patterns of rapid ATP depletion and AMP accumulation were similar to those found in rat. In rat liver, enzymatic regulatory control of glycolysis appeared to be extremely sensitive to the decline in cellular energy levels. After 8 h cold hypoxia levels of fructose-6-phosphate decreased and fructose-1,6-bisphosphate increased, thus reflecting an activation of glycolysis at the regulatory step catalysed by phospho-fructokinase fructose-1,6-bisphosphatase. Despite an initial increase in flux through glycolysis over the first 2 min (lactate levels increased 3.7 mol·g^-1), further flux through the pathway was not permitted even though glycolysis was activated at the phosphofructokinase/fructose-1,6-bisphosphatase locus at 8 h, since supplies of phosphorylated substrate glucose-1-phosphate or glucose-6-phosphate remained low throughout the duration of the 24-h period. Conversely, livers of Columbian ground squirrels exhibited no activation or inactivation of two key glycolytic regulatory loci, phosphofructokinase/fructose-1,6-bisphosphatase and pyruvate kinase/phosphoenolpyruvate carboxykinase and pyruvate carboxylase. Although previous studies have shown similar allosteric sensitivities to adenylates to rat liver phospho-fructokinase, there was no evidence of an activation of the pathway as a result of decreasing high energy adenylate, ATP or increasing AMP levels. The lack of any apparent regulatory control of glycosis during cold hypoxia may be related to hibernator-specific metabolic adaptations that are key to the survival of hypothermia during natural bouts of hibernation.Abbreviations DHAP dihydroxyacetonephosphate - EC energy charge - F1,6P₂ fructose-1,6-bisphosphate - F2,6P₂ fructose-2,6-bisphosphate - F6P fructose-6-phosphate - FBP fructose-1,6-bisphosphatase - G1P glucose-1-phosphate - G6P glucose-6-phosphate - GAP glyceraldehyde-3-phosphate - GAPDH glyceraldehyde-3-phosphate dehydrogenase - L/R lactobionate/raffinose-based solution - MR metabolic rate - PDH pyruvate dehydrogenase - PEP phosphoenolpyruvate - PEPCK & PC phosphoenolpyruvate carboxykinase and pyruvate carboxylase - PFK phosphofructokinase; PK, pyruvate kinase - Q ₁₀ the effect of a 10 °C drop in temperature on reaction rates (generally, Q ₁₀=2–3) - TA total adenylates - UW solution University of Wisconsin solution (L/R-based)     

Transformation of Atriplex gmelini plants from callus lines using Agrobacterium tumefaciens

Atriplex gmelini plants were regenerated via organogensis from hypocotyl explants. Callus lines were induced from the hypocotyl explants on Linsmaier and Skoog (LS) medium supplemented with 1 M benzyladenine and 5 M -naphthaleneacetic acid in the dark. Shoots were regenerated from the callus lines on LS medium supplemented with 20 M thidiazuron and 0.1 M -naphthaleneacetic acid under a high-intensity light condition (450 mol m^–2 s^–1). The regenerated shoots were rooted on LS medium without growth regulators to obtain fully developed plants. We succeeded in transforming Atriplex gmelini from callus lines using Agrobacterium tumefaciens.     

Locomotion of the filiform sperm of Littorina (Gastropoda,Prosobranchia)

Summary The filiform sperm of Littorina sitkana swims effectively in sea water and more viscous fluids, overcoming the problems of a non-uniform flagellar beat with an unusual mechanism, which involves three main events: (1) the sperm rotates anti-clockwise (looking from tail to head); then (2) stops rotating and stiffens itself to form a screw-shape, with the tail being held perpendicular to the middle piece, and finally; (3) reverses its rotation and propels itself forward in a clockwise spiral. The average velocity of sperm is approximately 185 ms with a rotational frequency of 24 revs. The mechanism of propulsion may involve two kinetic centers at opposite ends of the sperm, which coordinate their movements to produce anti-clockwise rotation, stationary twisting, or clockwise rotation, in a manner reminiscent of spirochaetes. Littorina sperm also exhibit slower methods of propulsion including swimming backwards (tail first) at 18 m, and gliding at about 30 m.The adaptive significance of the rapid propulsion is not obvious, because Littorina copulate and fertilize internally and at each stage in the transfer there are external aids to sperm transport, such as ciliary action (oviduct) and muscular expulsion (bursa and seminal receptacle). The filiform shape, however, is well-adapted for long-term storage in the female body. These points are discussed.     

Plant regeneration from callus and explants of Sesbania spp.

Root, hypocotyl and cotyledon explants of Sesbania bispinosa, Sesbania cannabina, Sesbania formosa, and Sesbania sesban were cultured on Murashige and Skoog medium with benzyladenine (BA; 2.22, 4.44, 8.88 M) in combination with 2,4-dichlorophenoxyacetic acid (2,4-d; 2.26, 4.52, 9.05 M), indolebutyric acid (IBA; 0.25, 0.49, 4.92 M) or naphthaleneacetic acid (NAA; 2.69, 5.37, 10.74 M). Although all explant types developed some callus, callus occurred earliest and continued to grow fastest with hypocotyls. Media including 2.4-d or NAA gave the fastest growing callus. Callus was subcultured up to 10 times at 20-day intervals and retained a rapid growth rate. Shoots regenerated readily from both hypocotyls or cotyledons but not from roots. Shoot organogenesis was most frequent with IBA (0.25–4.92 M) in combination with BA (4.44–8.88 M) and did not occur with 2,4-d. With each species at least one medium induced shoot differentiation from more than 50 percent of the callus pieces. With one exception, media containing IBA that induced shoot organogenesis on explants also did so in callus, but media containing NAA, even when effective with explants, did not cause differentiation of callus. Shoots that differentiated were excised and cultured on MS medium without growth regulators or with IBA (2.46, 4.92, 9.84 M). Roots developed after 3–8 days on an appropriate rooting medium, often without IBA. Rooted plantlets were transplanted to pots in a greenhouse and developed into normal plants. Suitable media and protocols for initiating and subculturing callus and regenerating whole plants in vitro from callus and explants have thus been established for four species of Sesbania.     

Effect of aluminium on yield and plant chemical concentrations of some temperate legumes

The aluminium (Al) tolerance of 34 temperate legume species (143 genotypes, including 57 from Trifolium repens) was determined in 60 experiments over a 3 year period in a low ionic strength (2.7 × 10^-3 M) solution culture. For each genotype, the relationship between solution Al³⁺ activity (M) and relative yield was determined and the Al³⁺ activity associated with a 50% reduction in yield (Al_RY50) calculated. In addition, plant chemical concentrations were determined in at least one genotype from most species. For white clover, Al_RY50 over all genotypes had an approximately normal distribution with mean of 1.31 M for the tops and 1.51 M for the roots, and a standard deviation of about 0.4. This suggested that Al tolerance had a polygenic inheritance. For the other species tested, Al_RY50 ranged from 0.15 to 4.53 M in the tops and from 0.21 to 4.89 M in the roots. In the tops and roots, 37% and 26% respectively of the genotypes had an Al_RY50 less than 1 M, including all species tested in the genera Melilotus and Medicago. Only 8% or 23% of the genotypes, based on the tops and roots respectively, had an Al_RY50 greater than 2, including all genotypes in the species Lotus pedunculatus. Except for Lotus, there were no consistent differences between genera in plant chemical concentrations. In Lotus, concentrations of Ca, Zn, Mn and Cu in the tops and of all elements except B in the roots were lower than that of the other species. The Al_RY50 of the species was not related to plant chemical concentrations in the absence of Al. Depending on the plant element, increasing solution Al concentrations had no significant effect on plant chemical concentrations for 56–94% of the species. When a significant effect did occur, increasing Al in solution generally decreased S and K concentrations and increased Mn, Zn, Cu Fe, B and Al concentrations in the tops and roots and decreased Ca concentrations in the tops. Plant P concentrations decreased in the tops but increased in the roots. Increasing Al in solution increase plant Al at the average rate of 44 g g^-1 M ^-1 (range 20–87) in the tops and 333 g M ^-1 (range 162–616) in the roots.     

Age dependency of worker bee response to queen pheromone in a four-armed olfactometer

Summary Behavioral responses of differently aged worker beesApis mellifera to a queen pheromonal extract were analysed. The bees were tested individually in a four-armed olfactometer, one arm being scented with the pheromonal extract. This extract was prepared from heads of 14–17-day-old unmated queens. Among the components of the blend, 470 g 9-keto-2-(E)-decenoic acid, 200 g 9-hydroxy-2-(E)-decenoic acid and 5 gp-hydroxybenzoic acid methyl ester per queen equivalent were dosed. An age dependency in the worker bees' olfactory response to the components of the queen extract was shown, the strongest response occurring below the age of 5 days.     

Direct regeneration of Drosera from leaf explants and shoot tips

An efficient protocol for the micropropagation of Drosera anglica, D. binata and D. cuneifolia is described. Proliferation was obtained from leaf segments and shoot tips, which served as initial explants. The regeneration capacity of explants was influenced by factors such as nutrient media, concentrations of growth regulators and the type of medium (liquid or solid). The highest number of plants regenerating from D. binata explants was obtained on the growth regulator-free Vacin and Went medium. In the case of D. anglica the highest proliferation rate was obtained on the Fast medium supplemented with 0.05 M 6-benzyladenine (BA) and 0.005 M -naphthaleneacetic acid (NAA), whereas for D. cuneifolia the optimal regeneration medium proved to be 1/2 MS with the growth regulator supplementation estimated at 0.2 M BA and 0.2 M NAA. Liquid media significantly increased the regeneration potential of D. anglica and D. binata explants.     

Rapid clonal propagation of Dioscorea zingiberensis

A protocol was developed for rapid in vitro propagation of Dioscorea zingiberensis Wright using stems as explants. MS medium with the macroelements at half strength and supplemented with 20.0 g l^–1 sucrose and 8.0 g l^–1 agar was used as basal medium. Lateral buds on nodal cuttings grew into shoots within 20 days after culture on basal medium supplemented with 4.4 M 6-benzylaminopurine (BAP) and 1.1 M -naphthalene acetic acid (NAA). The shoots were cut into segments and cultured on medium with 8.9 M BA and 5.4 M NAA for 30 days for callus formation. The callus was cut into pieces and cultured on medium containing 22.2 M BAP and 1.1 M NAA, on which 87.5% of the callus pieces regenerated multiple shoots within 50 days. The shoots were rooted on medium containing 4.9 M indole-3-butyric acid (IBA) for 20 days. Adventitious buds and shoots could be repeatedly formed after the calli were cut into pieces and cultured on the medium containing 8.9 M BAP plus 1.1 M NAA. More than 85% of the regenerated plantlets survived and grew vigorously 1 month after they were transplanted in vermiculite and each plant formed 2–4 microtubers 3 months of transplanting.     

In vitro culture of Aloe Barbadensis Mill.: Micropropagation from vegetative meristems

A method for rapid and highly effective plant micropropagation from vegetative meristems was established for Aloe barbadensis Mill. Plant micropropagation was achieved culturing apices on medium containing 1.1 M 2,4-dichlorophenoxyacetic acid and 2.3 M kinetin for 15–30 days. High morphogenetic ability was maintained by transferring explants (after 60 days) on media containing 0.11 M 2,4-dichlorophenoxyacetic acid and 2.2 M 6-benzylaminopurine.     

Plant regeneration from cotyledons of niger

Callus initiation from seedling explants of niger (Guizotia abyssinica Cass) cv. Ootacamund was found to be better on LS medium containing kinetin (1.4 M) plus 2,4-dichlorophenoxyacetic acid (9 M) than its analogues. Embryoids were induced directly from cotyledons on LS medium supplemented with 2,4,5-trichlorophenoxyacetic acid and 2,4,5-trichlorophenoxypropionic acid. When cotyledon-derived callus was subcultured onto medium with 10.7 M naphthalene-acetic acid and 2.3 M kinetin, embryogenesis was observed. Multiple shoots were obtained from cotyledonary explants in presence of MS medium containing 4.4 M benzyladenine and 11.4 M indoleacetic acid. Regenerated plants that were transferred to pots and grown to maturity were morphologically normal and fertile.Abbreviations NAA naphthaleneacetic acid - IAA indoleacetic acid - BA benzyladenine - GA₃ gibberellic acid - 2,4-D 2,4-dichlorophenoxyacetic acid - 2,4,5-T 2,4,5-trichlorophenoxypropionic acid - 2,4,5-TP 2,4,5-trichlorophenoxypropionic acid - ABA abscisic acid     

Substrate specificity and product inhibition of different forms of fructokinases and hexokinases in developing potato tubers

The substrate dependence and product inhibition of three different fructokinases and three different hexokinases from growing potato (Solanum tuberosum L.) tubers was investigated. The tubers contained three specific fructokinases (FK1, FK2, FK3) which had a high affinity for fructose K _m=64, 90 and 100 (M) and effectively no activity with glucose or other hexose sugars. The affinity for ATP (K _m=26, 25 and 240 M) was at least tenfold higher than for other nucleoside triphosphates. All three fructokinases showed product inhibition by high fructose (K _i=5.7, 6.0 and 21 mM) and were also inhibited by ADP competitively to ATP. Sensitivity to ADP was increased in the presence of high fructose, or fructose-6-phosphate. In certain conditions, the K _i (ADP) was about threefold below the K _m (ATP). All three fructokinase were also inhibited by fructose-6-phosphate acting non-competitively to fructose (K _i=1.3 mM for FK2). FK1 and FK2 showed very similar kinetic properties whereas FK3, which is only present at low activities in the tuber but high activities in the leaf, had a generally lower affinity for ATP, and lower sensitivity to inhibition by ADP and fructose. The tuber also contained three hexokinases (HK1, HK2, HK3) which had a high affinity for glucose (K _m=41, 130 and 35 M) and mannose but a poor affinity for fructose (K _m=11, 22 and 9 mM). All three hexokinases had a tenfold higher affinity for ATP (K _m=90, 280 and 560 M) than for other nucleoside triphosphates. HK1 and HK2 were both inhibited by ADP (K _i=40 and 108 M) acting competitively to ATP. HK1, but not HK2, was inhibited by glucose-6-phosphate, which acted non-competitively to glucose (K _i=4.1 mM). HK1 and HK2 differed, in that HK1 had a narrower pH optimum, a higher affinity for its substrate, and showed inhibition by glucose-6-phosphate. The relevance of these properties for the regulation of hexose metabolism in vivo is discussed.Abbreviations FK fructokinase - Fru6P fructose-6-phosphate - Glc6P glucose-6-phosphate - HK hexokinase - NTP nucleoside triphosphate - P_i inorganic phosphate - UDPGlc uridine-5-diphosphoglucose This work was supported by the Deutsche Froschungsgemeinschaft (SFB 137). We are grateful to Professor E. Beck (Lehrstuhl für Pflanzenphysiologie, Universität Bayreuth, FRG) for providing laboratory facilities.     

Secretion of Escherichia coli DsbA and DsbC proteins from Brevibacillus choshinensis: stimulation of human epidermal growth factor production

DsbA and DsbC, members of the thioredoxin super-family of redox proteins, which are expressed in the periplasmic space of Escherichia coli, were cloned into and successfully secreted from Brevibacillus choshinensis at 100 g ml^–1. Both proteins were active in exchanging disulfide bonds of bovine insulin in vitro. Furthermore, DsbA secreted by B. choshinensis promoted the conversion of non-native human epidermal growth factor to the native form.     

Ecotoxicity of lead to the zebra musselDreissena Polymorpha,Pallas

The effect of lead on the filtration rate of the zebra musselDreissena polymorpha was investigated, together with the accumulation of Pb in the soft tissues of the mussels. The NOEC-filtration was 116 g.l^–1 (0,56 mol.l^–1) and the EC₅₀-filtration was 370 g.l^–1 (1.79 mol.l^–1). The NOEC-accumulation was the concentration found in the control water (1.4g.l^–1). These experiments show that the EC₅₀-filtration for Pb is similar to that for Cd, higher than that for Cu and lower than that for Zn. The water quality criteria for lead allow 25 g Pb.l^–1 in surface water. This will not cause short-term effects. Long-term effects may, however, occur, since an accumulation of Pb as low as 16 g.l^–1 was recorded in this study.