The effect of Cd2+ on photosynthetic reactions of mesophyll protoplasts

Photosynthetic CO₂-fixation of mesophyll protoplasts of lambs lettuce [Valerianella locusta (L.) Betcke] was inhibited by short time exposure to Cd⁺. Inhibition was due to uptake of the metal ion into the protoplasts and increased with increasing Cd²⁺ concentrations and the time of preincubation. A 10 min pretreatment at 2 mM Cd²⁺ reduced CO₂-fixation by 40–60%. Inhibition of photosynthesis was independent of the light intensity to which the protoplasts were exposed. Measurement of the lightinduced electrochromic pigment absorption change at 518nm and chlorophyll fluorescence studies revealed that primary photochemical reactions associated with the thylakoid membranes were not affected by the metal ion. Also, light activation of the ribulose-1,5-bisphosphate carboxylase (EC 4.1.1.39) was not inhibited by Cd²⁺. Under rate-limiting CO₂ concentrations, inhibition of CO₂-fixation was smaller than at V_max of CO₂ reduction indicating that the carboxylation reaction of the Calvin cycle is not susceptible to Cd²⁺. Cd²⁺ treatment of protoplasts significantly extended the lagphase of CO₂-supported O₂-evolution and partly inhibited light activation of the glyceraldehyde-3-phosphate dehydrogenase (EC 1.2.1.13) and the ribulose-5-phosphate kinase (EC 2.7.1.19). Measurement of relative concentrations of [¹⁴C]-labeled Calvin cycle intermediates showed that Cd²⁺ caused a decrease in the 3-phosphoglycerate/triose phosphate ratio and an increase in the triose phosphate/ribulose-1,5-bisphosphate ratio. It is concluded that in protoplasts Cd²⁺ affects photosynthesis mainly at the level of dark reactions and that the site of inhibition may be localized in the regenerative phase of the Calvin cycle.     

Cadmium Uptake by Pinus resinosa Ait. Pollen and the Effect on Cation Release and Membrane Permeability

Cadmium uptake by red pine (Pinus resinosa Ait.) pollen from a graded series of Cd²⁺ solutions (0 to 2.88 microequivalents per 50 milligrams pollen) and its effect on membrane integrity were examined by atomic absorption spectroscopy. Uptake was strongly dependent on Cd²⁺ concentration and was limited to adsorption and cation exchange in pollen walls during a 3-hour measurement period. Good correlation between measured Cd²⁺ uptake and that predicted by the Langmuir and Freundlich isotherm equations indicated the adsorptive nature of Cd²⁺ uptake. While substantial quantities of Ca²⁺ and Mg²⁺ were released by exchange mechanisms concurrent with Cd²⁺ uptake, there was no evidence for leakage of cations due to membrane impairment as indicated by a poor correlation between Cd²⁺ uptake and K⁺ efflux. Virtually all Cd²⁺ removed from solution was freely exchangeable with 0.5 millimolar CaCl₂ and demonstrated that Cd²⁺ did not readily enter pine pollen but was adsorbed on the pollen wall. Ultraviolet transmission spectra of treatment solutions and analyses of phosphate and reducing sugar efflux also indicated that the potent toxicity of Cd²⁺ to pollen germination and germ tube elongation was not the result of membrane damage.     

Effects of the glutamine synthetase inhibitor methionine sulfoximine on CO2 fixation inLemna gibba

Summary Net CO₂ fixation inLemna gibba L. was inhibited by 0.5 mM L-methionine D,L-sulfoximine (MSX) both under photorespiratory conditions (21% O₂) and in 2% O₂. The inhibition was noticeably delayed by addition of 5 mM glutamine. Glutamine also delayed MSX-induced inactivation of glutamine synthetase. An increase in intracellular NH ₄ ⁺ concentration was noted in the presence of MSX only, and in the presence of 10 mM NH ₄ ⁺ only. However, presence of 10 mM NH ₄ ⁺ did not cause any inhibition of CO₂ fixation.     

Changes in dormancy of Sisymbrium officinaleseeds do not depend on changes in respiratory activity

Effects of dark incubation at different temperatures were studied on dormancy and respiratory activity of seeds of Sisymbrium officinale (L.) Scop. Because germination of this species absolutely depends on the simultaneous action of light and nitrate, changes in dormancy could be studied in darkness without the interference of early germination events. Upon the start of incubation rates of O₂ uptake and CO₂ release rose. This was followed by a gradual decrease until stable levels of O₂ uptake and CO₂ release were achieved. Seeds kept for prolonged periods at 24^°C, showed neither a change in germination capacity nor in rates of O₂ uptake and CO₂ release. Respiratory quotients were 0.55–0.7. The initial rise in O₂ uptake correlated with the rate of water uptake and with breaking of primary dormancy. However, the subsequent decline in O₂ uptake was not generally linked to induction of secondary dormancy. An increased O₂ uptake was not required during breaking of secondary dormancy. It is concluded that changes in dormancy are not generally related to changes in respiratory activity. However, germination strongly depends on respiration. The increase in O₂ uptake started well before radicle protrusion. A far red irradiation only reversed this increase when it was given before germination escaped from its red light antagonising action. The contribution of different respiratory pathways was followed during prolonged incubation at 24^°C in darkness. KCN at 1.5 mM was needed to inhibit the cytochrome pathway (CP) and benzohydroxamic acid (BHAM) at 30 mM to inhibit the alternative pathway (AP). These concentrations did not exert any side effects. Electron flow was predominantly via the CP, maximally 10% was via the AP. Flow through the CP declined during the first 6 days and residual respiration remained constant. Therefore, the contribution of residual respiration became relatively more important with prolonged incubation. KCN at concentrations that almost completely inhibited flow through the CP, did not dramatically reduce germination. BHAM already inhibited germination at concentrations that do not inhibit oxygen uptake.     

Effect of Cl on Cd uptake by Swiss chard in nutrient solutions

Swiss chard (Beta vulgaris L., cv. Fordhook Giant) was grown in nutrient solution with Cl concentrations varying between 0.01 mM and 120 mM. Solution Na concentration and ionic strength were maintained in all treatments by compensating with NaNO₃. All solutions contained Cd (50 nM, spiked with ¹⁰⁹Cd). Three different Cd²⁺ buffering systems were used. In one experiment, Cd²⁺ activity was unbuffered; its activity decreased with increased Cl concentration as a result of the formation of CdCl_n ^2–n species. In the other experiments, Cd²⁺ activity was buffered by the chelator nitrilotriacetate (NTA, 50 M) and ethylene-bis-(oxyethylenenitrilo)-tetraacetate (EGTA, 50 M) at about 10^–9 M and 10^–11 M, respectively. Plant growth was generally unaffected by increasing Cl concentrations in the three experiments. In unbuffered solutions, Cd concentrations in plant tissue decreased significantly (p<0.01) (approximately 2.4-fold) as solution Cl concentration increased from 0.01 mM to 120 mM. However, this decrease was smaller in magnitude than the 4.7-fold decrease in Cd²⁺ activity as calculated by the GEOCHEM-PC program for the same range of Cl concentrations. In solutions where Cd²⁺ activity was buffered by NTA, Cd concentrations in plant tissue increased approximately 1.4-fold with increasing Cl concentration in solution, while the Cd²⁺ activity was calculated to decrease 1.3-fold. In solutions where Cd²⁺ activity was buffered by EGTA, Cd concentrations in the roots increased 1.3-fold with increasing Cl concentration in solution but there was no effect of Cl on shoot Cd concentrations. The data suggest that either CdCl_n ^2–nspecies can be taken up by plant roots or that Cl enhances uptake of Cd²⁺ through enhanced diffusion of the uncomplexed metal to uptake sites.Abbreviations DAS days after sowing - EGTA ethylene-bis-(oxyethylenenitrilo)-tetraacetate - HBED N,N-bis(2-hydroxybenzyl)-ethylenediamine-N,N-diacetate - NTA nitrilotriacetate     

Effects of the interaction between ozone and carbon dioxide on gas exchange,photosystem II and antioxidants in rice leaves

To understand the interactive effects of O₃ and CO₂ on rice leaves; gas exchange, chlorophyll (Chl) fluorescence, ascorbic acid and glutathione were examined under acute (5 h), combined exposures of O₃ (0, 0.1, or 0.3 cm³ m⁻³, expressed as O₀, O_0.1, or O_0.3, respectively), and CO₂ (400 or 800 cm³ m⁻³, expressed as C⁴⁰⁰ or C⁸⁰⁰, respectively) in natural-light gas-exposure chambers. The net photosynthetic rate (P _N), maximum (F_v/F_m) and operating (F_q′/F_m′) quantum efficiencies of photosystem II (PSII) in young (8^th) leaves decreased during O₃ exposure. However, these were ameliorated by C⁸⁰⁰ and fully recovered within 3 d in clean air (O⁰ + C⁴⁰⁰) except for the O^0.3 + C⁴⁰⁰ plants. The maximum PSII efficiency at 1,500 μmol m⁻² s⁻¹ PPFD (F_v′/F_m′) for the O^0.3 + C₄₀₀ plants decreased for all measurement times, likely because leaves with severely inhibited P _N also had a severely damaged PSII. The P _N of the flag (16^th) leaves at heading decreased under O₃ exposure, but the decline was smaller and the recovery was faster than that of the 8^th leaves. The F_q′/F_m′ of the flag leaves in the O^0.3 + C⁴⁰⁰ and O^0.3 + C⁸⁰⁰ plants decreased just after gas exposure, but the F_v/F_m was not affected. These effects indicate that elevated CO₂ interactively ameliorated the inhibition of photosynthesis induced by O₃ exposure. However, changes in antioxidant levels did not explain the above interaction.     

Actions of cadmium on basolateral plasma membrane proteins involved in calcium uptake by fish intestine

Summary The inhibition of Ca^2–-ATPase, (Na⁺+K⁺)-ATPase and Na⁺/Ca²⁺ exchange by Cd²⁺ was studied in fish intestinal basolateral plasma membrane preparations. ATP driven ⁴⁵Ca²⁺ uptake into inside-out membrane vesicles displayed a K _m for Ca²⁺ of 88±17 nm, and was extremely sensitive to Cd²⁺ with an IC₅₀ of 8.2±3.0 pM Cd²⁺, indicating an inhibition via the Ca²⁺ site. (Na⁺+K⁺)-ATPase activity was half-maximally inhibited by micromolar amounts of Cd²⁺, displaying an IC₅₀ of 2.6±0.6 m Cd²⁺. Cd²⁺ ions apparently compete for the Mg²⁺ site of the (Na^– +K⁺)-ATPase. The Na⁺/Ca²⁺ exchanger was inhibited by Cd²⁺ with an IC₅₀ of 73±11 nm. Cd²⁺ is a competitive inhibitor of the exchanger via an interaction with the Ca²⁺ site (K _i = 11 nm). Bepridil, a Na⁺ site specific inhibitor of Na⁺/Ca²⁺ exchange, induced an additional inhibition, but did not change the K _i of Cd²⁺. Also, Cd²⁺ is exchanged against Ca²⁺, albeit to a lesser extent than Ca²⁺. The exchanger is only partly blocked by the binding of Cd²⁺. In vivo cadmium that has entered the enterocyte may be shuttled across the basolateral plasma membrane by the Na⁺/Ca²⁺ exchanger. We conclude that intracellular Cd²⁺ ions will inhibit plasma membrane proteins predominantly via a specific interaction with divalent metal ion sites.We would like to thank Dr. D. Fackre (University of Alberta, Canada) for stimulating discussions and Mr. F.A.T. Spanings (University of Nijmegen, The Netherlands) for excellent fish husbandry. The fura-2 measurements of intracellular Ca²⁺ concentrations in tilapia enterocytes were carried out in the Department of Physiology, School of Medicine, University of Alberta, Edmonton, Alberta T6G 2H7, Canada. Th.J.M. Schoenmakers and G. Flik were supported by travel grants from the Foundation for Fundamental Biological Research (BION) and the Netherlands Organization for Scientific Research (NWO).     

Permeability and Respiratory Properties of Germinating Pollen

A study of permeability and respiration of germinating lily pollen (Lilium longi-florum, variety Ace) was conducted. Calcium was needed in the culture medium to maintain the integrity of the cell membranes. Carbohydrates leaked from pollen when calcium was absent, but little or no leakage occurred when calcium was present. A culture medium containing penlaerythritol was developed for this study. Pentaerythritol allowed normal germination and did not interfere with measurement of carbohydrates. Intact pollen grains possessed a β-fructofuranosidase external to the cell membrane which hydrolyzed sucrose in the culture medium. External α- or β-glucosidases were absent, although soluble α-glucosidase was present within the pollen. Oligomycin, an inhibitor of oxidative phosphorylation, eliminated the 3-phase patters of respiration associated with normal germination. O₂ uptake was inhibited by oligomycin while CO₂ output was stimulated. 2,4-Dinitrophenol, an uncoupler of oxidative phosphorylation, reversed the inhibition of O₂ uptake caused by oligomycin.     

Exogenous Abscisic Acid Alleviates Cadmium Toxicity by Restricting Cd2+ Influx in Populus euphratica Cells

Abscisic acid (ABA), a widely known phytohormone involved in the plant response to abiotic stress, plays a vital role in mitigating Cd²⁺ toxicity in herbaceous species. However, the role of ABA in ameliorating Cd²⁺ toxicity in woody species is largely unknown. In the present study, we investigated ABA restriction on Cd²⁺ uptake and the relevance to Cd²⁺ stress alleviation in Cd²⁺-hypersensitive Populus euphratica. ABA (5 μM) markedly improved cell viability and growth but reduced membrane permeability in CdCl₂ (100 μM)-stressed P. euphratica cells. Moreover, ABA significantly increased the activity of the antioxidant enzymes catalase (CAT), superoxide dismutase (SOD), and ascorbate peroxidase (APX), contributing to the scavenging of Cd²⁺-elicited H₂O₂ within P. euphratica cells during the period of CdCl₂ exposure (100 μM, 24–72 h). ABA alleviation of Cd²⁺ toxicity was mainly the result of ABA restriction of Cd²⁺ uptake under Cd²⁺ stress. Steady-state and transient flux recordings showed that ABA inhibited Cd²⁺ entry into Cd²⁺-shocked (100 μM, 30 min) and short-term-stressed P. euphratica cells (100 μM, 24–72 h). Non-invasive micro-test technique data showed that H₂O₂ (3 mM) stimulated the Cd²⁺-elicited Cd²⁺ influx but that the plasma membrane (PM) Ca²⁺ channel inhibitor LaCl₃ blocked it, suggesting that the Cd²⁺ influx was through PM Ca²⁺-permeable channels. These results suggested that ABA up-regulated antioxidant enzyme activity in Cd²⁺-stressed P. euphratica and that these enzymes scavenged the Cd²⁺-elicited H₂O₂ within cells. The entry of Cd²⁺ through the H₂O₂-mediated Ca²⁺-permeable channels was subsequently restricted; thus, Cd²⁺ buildup and toxicity were reduced in the Cd²⁺-hypersensitive species, P. euphratica.

     

ATP-driven Ca2+ transport in sealed plasma membrane vesicles prepared by aqueous two-phase partitioning from leaves of Commelina communis

Sealed plasma membrane vesicles were obtained in high purity from leaves of Commelina communis L. by aqueous two-phase partitioning. Based on the analysis of a range of markers, the preparations (U₃+U₃′ phases) were shown to be devoid of tonoplast, Golgi and thylakoid membranes, and showed only trace mitochondrial contamination. One-third of the vesicles were oriented inside out and exhibited ATP-driven ⁴⁵Ca²⁺ transport [? 15 pkat (mg protein)⁻¹]. Ca²⁺ uptake into the vesicles had a pH optimum of 7.2 and apparent K_m values for Ca²⁺ of 4.4 μM and for Mg-ATP of 300 μM. Ca²⁺ uptake, K⁺, Mg²⁺-ATPase (EC 3.6.1.3) activity as well as glucan synthase II (EC 2.4.1.34) activity were all maximal at the same equilibrium density (1.17 g cm⁻³) on continuous sucrose density gradients. The protonophore carbonylcyanide m-chlorophenylhydrazone (CCCP) did not inhibit the ATP-dependent Ca²⁺ transport into the vesicles, excluding a Ca²⁺/H⁺ exchange driven by a proton gradient. ATP-dependent Ca²⁺ uptake was inhibited by erythrosin B (I₅₀= 0.1 μM), ruthenium red (I₅₀= 30 μM), La³⁺ (I₅₀= 10 μM) and vanadate (I₅₀= 500 μM), but not by azide, cyanide and oligomycin. The calmodulin antagonists, trifluoperazine (I₅₀= 70 μM) and W-7 (I₅₀= 100 μM) were also inhibitory, However, this inhibition was not overcome by calmodulin. Trifluoperazine and W-7, on the other hand, stimulated Ca²⁺ efflux from the vesicles rather than inhibit Ca²⁺ uptake. Our results demonstrate the presence of a Ca²⁺-ATPase in the plasma membrane of C. communis. In the intact cell, the enzyme would pump Ca²⁺ out of the cell. Its high affinity for Ca²⁺ makes it a likely component involved in adjusting low cytoplasmic Ca²⁺ levels. No indications for a secondary active Ca²⁺/H⁺ transport mechanism in the plasma membrane of C. communis were obtained. Both, the nucleotide specificity and the sensitivity towards vanadate. distinguish the Ca²⁺-ATPase from the H⁺-translocating K⁺. Mg²⁺-ATPase in C. communis plasma membranes.     

Effects of exogenous glycinebetaine on growth, CO2 assimilation, and photosystem II photochemistry of maize plants

Effects of exogenous glycinebetaine (GB, 2–50 mM) on growth, photosynthetic gas exchange, PSII photochemistry, and the activities of key enzymes involved in CO₂ fixation in maize plants were investigated. Growth, CO₂ assimilation rate, and stomatal conductance increased at low GB concentrations (2–20 mM) but decreased significantly at high GB concentrations (30–50 mM). Leaf relative water content and water potential remained unchanged at low GB concentrations but decreased at high GB concentrations. The maximal efficiency of PSII photochemistry was unchanged either at low or high GB concentrations. The actual PSII efficiency ( Φ _PSII) and photochemical quenching (q_P) increased at low GB concentrations but decreased at high GB concentrations. At low GB concentrations, there were no significant changes in the efficiency of excitation energy capture by open PSII reaction centres (F_v′/F_m′) and non‐photochemical quenching (q_N). At high GB concentrations, F_v′/F_m′ decreased while q_N increased significantly. There were no changes in the activities of phosphoenolpyruvate carboxylase, pyruvate phosphate dikinase, and ribulose‐1,5‐bisphosphate carboxylase in control and GB‐fed plants. However, there was a linear correlation between CO₂ assimilation rate and stomatal conductance in control and GB‐fed plants. Moreover, there were no significant differences in O₂ evolution rate between control and GB fed‐plants under saturated CO₂ conditions. The results suggest that exogenous GB application at certain concentrations can enhance CO₂ assimilation rate, which can be explained by an increased stomatal conductance.     

Effect of SO32- on the activity of ribulose bisphosphate carboxylase from seedlings of Pinus silvestris

Abstract The effect of sulphite on ribulose bisphosphate carboxylase, extracted from needles of Pinus silvestris L., was studied in vitro at pH 8.15 and 25°C. 1 mM and higher concentrations of SO₃^2- inhibited the enzyme. The enzyme was activated either in the assay medium (2.5 – 20 mM HCO_3, 20 mM MgCl₂) or in 10 or 20 mM HCO₃^- and 20–25 mM MgCl₂. Linear reciprocal plots of the activity versus the substrate concentration were obtained, when the HCO₃^- concentration during activation was 4 mM or higher. When the enzyme was activated at high HCO₃^- and Mg²⁺ concentrations, the K_m(CO₂) was c. 27 μM. With respect to HCO₃^-. SO₃^2- inhibited the enzyme in a non-competitive fashion. The inhibition was similar, whether SO₃^2- was present during activation or not. Apparently. SO₃^2- did not interfere with the binding of CO₂ and Mg²⁺ at the activating site. The K₁ was 11–13 mM SO₃^2-. With respect to ribulose bisphosphate the inhibition was also noncompetitive. Similar results with respect to HCO₃^- were obtained for spinach, Spinacia oleracea L., which is contrary to earlier reports.     

Cadmium exerts its toxic effects on photosynthesis via a cascade mechanism in the cyanobacterium,Synechocystis PCC 6803

Despite intense research, the mechanism of Cd²⁺ toxicity on photosynthesis is still elusive because of the multiplicity of the inhibitory effects and different barriers in plants. The quick Cd²⁺ uptake in Synechocystis PCC 6803 permits the direct interaction of cadmium with the photosynthetic machinery and allows the distinction between primary and secondary effects. We show that the CO₂‐dependent electron transport is rapidly inhibited upon exposing the cells to 40 µm Cd²⁺ (50% inhibition in ～15 min). However, during this time we observe only symptoms of photosystem I acceptor side limitation and a build of an excitation pressure on the reaction centres, as indicated by light‐induced P700 redox transients, O₂ polarography and changes in chlorophyll a fluorescence parameters. Inhibitory effects on photosystem II electron transport and the degradation of the reaction centre protein D1 can only be observed after several hours, and only in the light, as revealed by chlorophyll a fluorescence transients, thermoluminescence and immunoblotting. Despite the marked differences in the manifestations of these short‐ and long‐term effects, they exhibit virtually the same Cd²⁺ concentration dependence. These data strongly suggest a cascade mechanism of the toxic effect, with a primary effect in the dark reactions.     

Application of capillary gas chromatography to the study of hydrolysis of the nerve agent VX in rat plasma

We present here a gas chromatography technique allowing the detection and quantification of VX [O-ethyl S-(2-diisopropylaminoethyl) methylphosphonothiolate] a as well as its P---S bond hydrolysis product diisopropylaminoethanethiol directly from spiked rat plasma. This technique was applied to study VX hydrolysis in rat plasma. We observed that 53±4% of 374 μM VX disappeared from spiked plasma after 2 h. VX disappearance was mainly related to enzymatic cleavage of the P---S bond (K_m=2.5 mM and V_max=13.3 nmol min⁻¹ of rat plasma). The activity was totally inhibited by 1 mM Hg²⁺ and was also inhibited by metal chelators.     

Effects of Various Concentrations of Carbon Dioxide on Respiration and Potassium Uptake in Barley Roots

Barley roots contain a CO₂ sensitive respiratory fraction which is inhibited in 50 per cent CO₂ and is partially restored upon subsequent exposure to air. The residual O₂ consumption occurring at CO₂ concentrations between 50 per cent and 95 per cent amounts to about 40 per cent of the O₂ uptake in air and can support K⁺ uptake for a limited time at a rate equal to or higher than occurs in air. Above 95 per cent CO₂ both O₂ and K⁺ uptakes decrease rapidly. 2,4-dinitrophenol (DNP), in the range of 10^?6 to 10^?5M, stimulates O₂ uptake by the roots in air. The stimulation is absent when roots are treated with DNP in 80 per cent CO₂, presumably because of the reduced demand for inorganic phosphate and phosphate acceptor at the lower respiratory level in high CO₂. In either air or CO₂, K⁺ uptake is strongly inhibited by DNP. A comparison of the respiratory and K⁺ uptake data indicates that O₂ consumption is a necessary requirement for the uptake process in high CO₂. Protoplasmic streaming in the root cells is rapidly stopped by high CO₂ although K⁺ uptake and O₂ consumption continue. The cation uptake mechanism in high CO₂ appears to be limited to the stationary cytoplasm. It is also possible that a similar mechanism may be involved in cation uptake in air.     

Oxygen Uptake and Photosynthesis of the Red Macroalga, Chondrus crispus, in Seawater: Effects of Light and CO(2) Concentration

With an experimental system using mass spectrometry techniques and infra-red gas analysis of CO₂ developed for aquatic plants, we studied the responses to various light intensities and CO₂ concentrations of photosynthesis and O₂ uptake of the red macroalga Chondrus crispus S. The CO₂ exchange resistance at air-water interface which could limit the photosynthesis was experimentally measured. It allowed the calculation of the free dissolved CO₂ concentration. The response to light showed a small O₂ uptake (37% of net photosynthesis in standard conditions) compared to C₃ plants; it was always higher than dark respiration and probably included a photoindependent part. The response to CO₂ showed: (a) an O₂ uptake relatively insensitive to CO₂ concentration and not completely inhibited with high CO₂, (b) a general inhibition of gas exchanges below 130 microliters CO₂ per liter (gas phase), (c) an absence of an inverse relationship between O₂ and CO₂ uptakes, and (d) a low apparent K_m of photosynthesis for free CO₂ (1 micromolar). These results suggest that O₂ uptake in the light is the sum of different oxidation processes such as the glycolate pathway, the Mehler reaction, and mitochondrial respiration. The high affinity for CO₂ is discussed in relation to the use of HCO₃⁻ and/or the internal CO₂ accumulation.     

Effects of Monovalent and Divalent Cations on Ca2+ Fluxes Across Chromaffin Secretory Membrane Vesicles

Abstract: Bovine chromaffin secretory vesicle ghosts loaded with Na⁺ were found to take up Ca²⁺ when incubated in K⁺ media or in sucrose media containing micromolar concentrations of free Ca²⁺. Li⁺- or choline⁺loaded ghosts did not take up Ca²⁺. The Ca²⁺ accumulated by Na⁺-loaded ghosts could be released by the Ca²⁺ ionophore A23187, but not by EGTA. Ca²⁺ uptake was inhibited by external Sr²⁺, Na ⁺, Li ⁺, or choline ⁺. All the ⁴⁵Ca²⁺ accumulated by Na⁺-dependent Ca²⁺ uptake could be released by external Na ⁺, indicating that both Ca²⁺ influx and efflux occur in a Na⁺-dependent manner. Na ⁺ -dependent Ca²⁺ uptake and release were only slightly inhibited by Mg²⁺. In the presence of the Na⁺ ionophore Monensin the Ca²⁺ uptake by Na ⁺-loaded ghosts was reduced. Ca²⁺ sequestered by the Na⁺-dependent mechanism could also be released by external Ca²⁺ or Sr²⁺ but not by Mg²⁺, indicating the presence of a Ca²⁺/Ca²⁺ exchange activity in secretory membrane vesicles. This Ca²⁺/Ca²⁺ exchange system is inhibited by Mg²⁺, but not by Sr²⁺. The Na ⁺ -dependent Ca²⁺ uptake system in the presence of Mg²⁺ is a saturable process with an apparent K_m of 0.28 μM and a V_max= 14.5 nmol min^?1 mg protein^?1. Ruthenium red inhibited neither the Na⁺/Ca²⁺ nor the Ca²⁺/Ca²⁺ exchange, even at high concentrations.     

Purification and Characterization of a Phytase from <Emphasis Type="Italic">Pseudomonas syringae</Emphasis> MOK1

A phytase (EC 3.1.3.8) from Pseudomonas syringae MOK1 was purified to apparent homogeneity in two steps employing cation and an anion exchange chromatography. The molecular weight of the purified enzyme was estimated to be 45 kDa by sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis. The optimal activity occurred at pH 5.5 and 40°C. The Michaelis constant (K _m ) and maximum reaction rate (V_max) for sodium phytate were 0.38 mM and 769 U/mg of protein, respectively. The enzyme was strongly inhibited by Cu²⁺, Cd²⁺, Mn²⁺, and ethylenediaminetetraacetic acid (EDTA). It showed a high substrate specificity for sodium phytate with little or no activity on other phosphate conjugates. The enzyme efficiently released orthophosphate from wheat bran and soybean meal.Received: 9 September 2002 / Accepted: 6 December 2002     

CO2 and O2 gas exchange in outdoor thin-layer high density microalgal cultures

Two thin layer culture units operated as batch cultures with the algaChlorella kessleri were used in gas exchange experiments. The mass transfer coefficient K_g [g m^–2 h^–1 kPa^–1] of O₂ and CO₂ desorption from culture surface decreased with increasing culture temperature. Between 60–70% of supplied CO₂ was used for algal growth. It was estimated that the length of growth surface may be extended to about 50 m, without additional saturation by CO₂. On average 1.35 g CO₂ was consumed by the alga per 1 g of produced O₂. Net CO₂ consumption (R_CO2) and O₂ production (R_O2) were not inhibited by irradiance. R_O2 did not decrease (in some cases it even increased) along the culture surface, despite increased accumulation of O₂. Measurement of pO₂ where the culture leaves the reactor before being pumped back onto the illuminated surface, correlated with O₂ production and CO₂ consumption and may be used to monitor the reactors growth performance.     

The involvement of hydrogen peroxide in UV-B-inhibited pollen germination and tube growth of Paeonia suffruticosa and Paulownia tomentosa in vitro

Previous studies have shown that UV-B could affect pollen germination and tube growth. However, the mechanism of response of pollen to UV-B has not been clear. The purpose of this study was to investigate the role of hydrogen peroxide (H₂O₂) in the UV-B-induced reduction of in vitro pollen germination and tube growth of Paeonia suffruticosa Andr. and Paulownia tomentosa Steud. Exposure of pollen of the two species to 0.4 and 0.8 W m⁻² UV-B radiation for 3 h resulted in not only the reduction of pollen germination and tube growth, but also the H₂O₂ production in pollen grain and tube. Also, exogenous H₂O₂ inhibited pollen germination and tube growth of the two species in a dose-dependence manner. Two scavengers of H₂O₂, ascorbic acid and catalase, largely prevented not only the H₂O₂ generation, but also the reduction of pollen germination and tube growth induced by UV-B radiation in the two species. These results indicate that H₂O₂ is involved in the UV-B-inhibited pollen germination and tube growth.