Optimization of a pseudo-affinity process for penicillin acylase purification

A pseudo-affinity process for penicillin acylase (EC 3.5.1.11) purification using an affinity ligand (Ampicillin) attached on Sepharose 4B-CNBr was optimized. The enzyme adsorption on this affiant (Amp-Seph) is independent of pH between 5.5 and 8.8, in 100?mM phosphate containing 22% (w/v) ammonium sulphate. The desorption of the penicillin acylase from the affinity gels was carried out, the best desorption results being obtained through a non specific eluent, 100?mM phosphate pH 4.6 with 15% (w/v) ammonium sulphate. The best purification results were obtained with an enzymatic extract, produced through osmotic shock of Escherichia coli cells (3.7?IU/mg prot). With this extract and an affinity gel of Sepharose 4B-CNBr derivatized with ampicillin (3.8?μmol/cm³?gel), a maximum activity capacity adsorbed of 20?IU/cm³?gel was obtained for initial values of activity and protein concentration of 1.7?IU/cm³ and 0.4?mg prot/cm³, respectively. With the optimized eluent it was possible to obtain penicillin acylase in only one purification step with a desorption yield of enzyme activity higher than 90%. The penicillin acylase produced with this process was characterized by a maximum purity of 34?IU/mg prot, corresponding to a purification degree higher than 150 in relation to the lowest pure enzymatic extract. The enzyme purity of the eluted fractions was certified by SDS gel electrophoresis and liquid chromatography through a Mono Q column in a FPLC apparatus. The gel electrophoresis presented 4 main stained bands with 2 corresponding to α and β subunits of the penicillin acylase with equivalent molecular weights of 27 and 63?kDa. No external diffusion resistance on penicillin acylase and total protein adsorption on this affiant (Amp-Seph 3.8?μmol/cm³?gel) were observed for continuous adsorption processes performed at two different agitation speeds (120 and 400?rpm).     

Bt毒素在三种矿物表面的吸附与解吸

研究了Bt库斯塔克亚种(kurstaki）毒素（65 kDa）在高岭土、针铁矿和氧化硅表面的吸附和解吸特性.结果表明：在磷酸盐缓冲体系（pH 8）中,3种矿物的等温吸附曲线均符合Langmuir方程（R²>0.9661）,它们对Bt毒素的吸附顺序为：针铁矿﹥高岭土﹥二氧化硅.矿物对Bt毒素的吸附1 h就基本达到了吸附平衡.在pH 6～8范围内,针铁矿、高岭土和二氧化硅对Bt毒素的吸附量随pH值的升高而降低.10 ℃～50 ℃范围内,针铁矿和氧化硅对Bt毒素吸附量随温度升高有所下降（8.39％和47.06％）,高岭土对Bt毒素吸附则略有升高（5.91％）.红外光谱分析显示,Bt毒素被矿物吸附后结构仅有微小变化.被矿物吸附的Bt毒素不易被去离子水解吸,水洗3次总解吸率为28.48%～42.04%.     

Characterization of phosphate absorption in maize root cortex segments

Uptake of phosphate ions by 1 mm segments of isolated maize root cortex layers was studied. Cortex segments (from roots of 8 days old maize plants) absorb phosphate ions from 1 mM KH₂PO₄ in 0.2 mM CaSCO₄ at the average rate of 34.3 ±3.2 μg P_i g^?1 (fr. m.) h^?1,i.e. 0.35± 0.02 μmol P_i g^?1 (fr. m.) h^?1. Phosphate uptake considerably increases after a certain period of “augmentation”,i.e. washing in aerated 0.2 mM CaSO₄. This increase is completely blocked by the presence of 10 μg ml^?1 cycloheximide. The relation of uptake rate to phosphate concentration in the medium was shown to have 3 phases in the concentration range of 0.02 - 40 mM. Transition points were found between 0.8–1 mM and 10–20 mM. Following K_m and V_max values were found: K_m[mM] : 0.37 - 3.82 - 27.67 V_max[μg P_i g^?1 (fr. m.) h^?1] : 3.33 - 39.40 - 66.67 We have found no sharp pH optimum for phosphate uptake. It proceeds at almost constant rate till pH 6.0 and then the uptake rate drops with increasing pH. At low phosphate concentrations (1 mM) the lowest uptake rate was found at 5 and 13 °C, while the uptake is higher at 5 °C than at 13 °C at phosphate concentrations higher than 1 mM. At these concentrations uptake rate at 35 °C is lower than at 25 °C. Phosphate uptake considerably decreased in anaerobic conditions. DNP and iodoacetate (0.1 mM) completely blocked phosphate uptake from 1 mM KH₂PO₄, while uptake from 5 and 10 mM KH₂PO₄ was left unaffected by these substances. The inhibitors of active - SH groups NEM and PCMB inhibited phosphate uptake: 10^?3 M NEM by 81.6%, 10⁴ M NEM by 42% and 10^?4 M PCMB by 42%.     

The influence of phosphate concentration on the kinetics of uptake by maize roots

In an experiment with native maize roots depending on different phosphorus concentration in the external solution (0.001 … 50 mM P), the multiphasic character of the kinetics of phosphate uptake has been stated. The single phases are characterized by the different values of K_m and V_max. In the wide range of concentrations the isotherm of the phosphate uptake has five evident phases. The character of kinetics for the uptake of phosphate is analogical to the kinetics of the enzymatic reactions described by the Michaelis-Menten equation. On the other hand the linear dependence for the inactivated root was determined,i.e. the uptake of phosphate versus different phosphorus concentration in the external solution. The graphic representation of the logarithmic values for the phosphorus taken up versus the different phosphorus concentration in the external solution gives the biphasic course including concentration less than 1.0 mM P and more than 1.0 mM P. Within the framework of the concentration range the following values of V_max, K_m and ϕ_in were calculated under the conditions if the concentration of phosphorus is less than 1.0mMP: V_max = 1.705 μmol P × g^-1h^-1, K_m = 0.057 mM P and ϕ_in = 0.83,i.e. if the concentration of phosphorus is more than 1.0mM P: V_max = 40 μmol P × g^-1 h^-1, K_m = 16.66 mM and ϕ_in = 20. According to these results, the phosphate concentration in the external solution influences the activity of the transport mechanisms concerning their conformative changes which discretely change their working regime of membrane transport. This is also demonstrated in the change of values V_max, K_m and ϕ_in.     

Organic acid exudation and pH changes by Gordonia sp. and Pseudomonas fluorescens grown with P adsorbed to goethite

The actinomycete Gordonia sp. and the bacterium Pseudomonas fluorescens Pf-5 were grown in liquid media (pH 6.5) with phosphate adsorbed to the Fe-oxide/hydroxide goethite (Goe-P) and with soluble phosphate (0.1 mM or 1.0 mM P as KH2PO4). The two isolates showed distinct differences in their physiology. The pH of the medium was increased by Gordonia sp. by 1.1-1.7 units while it was decreased by P. fluorescens by 1.4-2.4 units. In all treatments the concentration of organic acids in the media with Gordonia sp. was up to 10 times lower (0.4-10.9 micromol L(-1)) than in media with P. fluorescens (33.4-84.4 micromol L(-1)). Gordonia sp. produced five different organic acids in varying amounts depending on P source and time. In contrast, P. fluorescens exuded mainly citrate and only small amounts of two to three other organic acids irrespective of P source or time.     

Phosphate adsorption kinetics of resuspended sediments in a shallow lake,Neusiedlersee, Austria

The phosphate adsorption capacity of Neusiedlersee suspended sediments was evaluated using a silicone oil-filter-centrifugation technique with³²P and³H-H₂O labelling. This technique is quick, convenient and allows uptake processes to be followed during the first few seconds. The time dependent phosphate uptake was tested for concentration and pH dependency; the highest uptake was recorded at pH 9.00. The high calcite proportion in the sestonic material seems to influence the adsorption process. The kinetics of adsorption (at lake orthophosphate level and pH) could be explained by a biphasic Langmuir isotherm.     

Contrasting responses of sulphate and phosphate transport in barley (Hordeum vulgare L.) roots to protein-modifying reagents and inhibition of protein synthesis

The uptake of sulphate into roots of barley seedlings is highly sensitive to phenylglyoxal (PhG), an arginine-binding reagent. Uptake was inhibited by >80% by a 1-h pre-treatment of roots with 0.45 mol · m^–3 PhG. Inhibition was maximal in pre-treatment solutions buffered between pH 4.5 and 6.5. Phosphate uptake, measured simultaneously by double-labelling uptake solutions with ³²P and ³⁵S, was less susceptible to inhibition by PhG, particularly at pH <6.5, and was completely insensitive to the less permeant reagent p-hydroxyphenylglyoxal (OH-PhG) administered at 1 mol · m^–3 at pH at 5.0 or 8.2; sulphate uptake was inhibited in -S plants by 90% by OH-PhG-treatment. Root respiration in young root segments was unaffected by OH-PhG pre-treatment for 1 h and inhibited by only 17% after 90 min pre-treatment. The uptake of both ions was inhibited by the dithiol-specific reagent, phenylarsine oxide even after short exposures (0.5–5.0 min). Sulphate uptake was more severely inhibited than that of phosphate, but in both cases inhibition could be substantially reversed by 5 min washing of treated roots by 5 mol · m^–3 dithioerythritol. After longer pre-treatment (50 min) with phenylarsine oxide, inhibition of the ion fluxes was not relieved by washing with dithioerythritol. Inhibition of sulphate influx by PhG was completely reversed by washing the roots for 24 h with culture solution lacking the inhibitor. The reversal was dependent on protein synthesis; less than 20% recovery was seen in the presence of 50 mmol · m^–3 cycloheximide. Sulphate uptake declined rapidly when -S roots were treated with cycloheximide. In the same roots the phosphate influx was little affected, small significant inhibitions being seen only after 4 h of treatment. Respiration was depressed by only 20% in apical and by 31% in basal root segments by cycloheximide pre-treatment for 2 h. Similar rates of collapse of the sulphate uptake and insensitivity of phosphate uptake were seen when protein synthesis was inhibited by azetidine carboxylic acid, p-fluorophenylalanine and puromycin. Considering the effects of all of the protein-synthesis inhibitors together leads to the conclusion that the sulphate transporter itself, or some essential sub-component of the uptake system, turns over rapidly with a half-time of about 2.5 h. The turnover of the phosphate transporter is evidently much slower. The results are discussed in relation to strategies for identifying the transport proteins and to the regulation of transporter activity during nutrient stress.Abbreviations CAP chloramphenicol - CHM cycloheximide - DTE dithioerythritol - OH-PhG p-hydroxyphenylglyoxal - PhAsO phenylarsine - PhG phenylglyoxal Paper dedicated to the memory of the late Ken Treharne who did much to encourage this collaboration.D.T.C. gratefully acknowledges a fellowship provided by Le Ministére des Etrangers during his stay in Montpellier.     

Effects of Temperature,pH and Salt Concentrations on the Adsorption of Bacillus subtilis on Soil Clay Minerals Investigated by Microcalorimetry

Adsorption of microorganisms on minerals is a ubiquitous interfacial phenomenon in soil. Knowledge of the extent and mechanisms of bacterial adsorption on minerals is of great agronomic and environmental importance. This study examined adsorption of Bacillus subtilis on three common minerals in soils such as kaolinite, montmorillonite and goethite under various environmental conditions. Isothermal titration calorimetry (ITC) was used to investigate the effects of temperature (20, 30, and 40°C), pH (5.0, 7.0, and 9.0) and KNO₃ concentration (0.001, 0.01, and 0.1 mol L^?1) on the adsorption by direct measurement of enthalpies. The results revealed that the adsorption process in all the mineral systems were exothermic, with the enthalpy changes (ΔH_ads ) ranging from ?52 to ?137, ?33 to ?147, and ?53 to ?141 kJ kg^?1 (dry weight of adsorbed bacteria) for kaolinite, montmorillonite, and goethite, respectively. No obvious dependence of ΔH_ads on temperature was observed. The heat release for all the systems generally declined with pH and decrease of salt concentration, which can be explained by the variations of hydrophobicity and electrostatic force with pH or salt concentration. The largest decrease was found for goethite among the three minerals from pH 5.0 to 7.0, suggesting that electrostatic attraction may play a more important role in bacterial adsorption on this mineral. The ΔH_ads values for all the minerals became nearly the same at pH 9.0, indicating that the same force probably hydrophobicity governing the adsorption for the minerals in alkaline environment. It is assumed that acidic or saline soils and the associated environments favor the adsorption of B. subtilis on clay minerals. In addition, the negative enthalpies expressed as kJ kg^?1 (carbon) revealed an energy flow into the environment accompanied by the carbon adsorption on the minerals in soil.     

Transport du sulfate à travers la double membrane limitante, ou enveloppe, des chloroplastes d'épinard

Evidence is presented for low rates of carriermediated uptake of sulphate, thiosulphate and sulphite into the stroma of the C3 plant Spinacia oleracea. Uptake of sulphate in the dark was followed using two techniques (1) uptake of sulphate [³⁵S] as determined by silicon oil centrifugal filtration and (2) uptake as indicated by inhibition of CO₂-dependant O₂ evolution rates after addition of sulphate.Sulphate, thiosulphate and sulphite were transported across the envelope leading to an accumulation in the chloroplasts. Sulphate transport had saturation kinetics of the Michaelis-Menten type (Vmax : 25 μmoles . mg⁻¹ chl . h⁻¹ at 22°C ; Km : 2.5 mM). The rate of transport for sulphate was not influenced either by illumination or pH change in the external medium. Phosphate was a competitive inhibitor of sulphate uptake by chloroplasts (Ki : 0.7 mM, fig. 1). The rate of transport for phosphate appeared to be much higher than for sulphate. When the chloroplasts were pre-loaded with labelled sulphate, radioactivity was rapidly released after addition of phosphate into the external medium. Consequently, the transport of sulphate occurs by a strict counter-exchange : for each molecule of sulphate entering the chloroplast, one molecule of phosphate leaves the stroma, and vice-versa.The uptake of sulphate by isolated intact chloroplasts exchanging for internal free phosphate induced a lower rate of photophosphorylation, which in turn inhibited CO₂-dependent O₂ evolution.The presence, on the inner membrane of the chloroplast envelope, of a specific sulphate carrier, distinct from the phosphate translocator, is discussed.     

Influence of phosphate status on phosphate uptake kinetics of maize (Zea mays) and soybean (Glycine max)

To obtain plants of different P status, maize and soybean seedlings were grown for several weeks in flowing nutrient solution culture with P concentrations ranging from 0.03–100 µmol P L^-1 kept constant within treatments. P uptake kinetics of the roots were then determined with intact plants in short-term experiments by monitoring P depletion of a 3.5 L volume of nutrient solution in contact with the roots. Results show maximum influx, I_max, 5-fold higher in plants which had been raised in solution of low compared with high P concentration. Because P concentrations in the plants were increased with increase in external P concentration, I_max was negatively related to % P in shoots. Michaelis constants, K_m, were also increased with increased pretreatment P concentration, only slightly with soybean, but by a factor of 3 with maize. The minimum P concentration, C_min, where net influx equals zero, was found between 0.06 and 0.3 µmol L^-1 with a tendency to increase with pretreatment P concentration. Filtration of solutions at the end of the depletion experiment showed that part of the external P was associated with solid particles.It was concluded that plants markedly adapt P uptake kinetics to their P status, essentially by the increase of I_max, when internal P concentration decreases. Changes of K_m and C_min were of minor importance.     

The reaction between some dyes and synthetic hydroxyapatite 2. Nature of the binding reaction

Synopsis In order to study the reactions involved in some of the histochemical procedures used for demonstrating calcium in calcified tissues, it was considered appropriate to use well characterized synthetic hydroxyapatite in the first instance. In the first paper of this series (Speirs, 1970), it was found that many dyes not previously used in histochemistry were capable of staining hydroxyapatite; the purpose of the present paper is to describe the numerous experimental approaches that have been made in an attempt to elucidate the mechanisms involved in the adsorption of some of these dyes by hydroxyapatite. Dyes have been grouped according to their adsorption curves (in which dye uptake by solid was plotted against the concentration of dye in solution at equilibrium). From these graphs, predictions and calculations were made concerning the orientation of the dye molecules on the surface of hydroxyapatite, the type of bonding possibly involved and the area of surface covered by each molecule. These were then related to the dimensions and structure of the dye molecules. Saturation of surface sites was achieved in the adsorption of some dyes and the nature of these sites was investigated by studying (1) competition between several dyes for the surface, (2) the accessibility of surface calcium and phosphorus in stained and unstained hydroxyapatite, and (3) the release of³²P from surface labelled hydroxyapatite during dye adsorption. Most of the dyes adsorbed from 95% ethanol were displaced relatively easily by treatment with 0.5 mM phosphate in ethanol, but those adsorbed from tris buffer, pH 7.45, were more stable when exposed to phosphate in tris. Treatment of stained hydroxyapatite with solvents containing 0.5 mM calcium reduced the rate of elution of the dyes. Convincing evidence for chelation, hydrogen bonding, ion exchange and physical adsorption processes as the mechanisms of adsorption has not been obtained. Future studies to investigate these processes are discussed.     

Effect of N source during soybean pod filling on nitrogen and sulfur assimilation and remobilization

During pod filling, a grain legume remobilizes vegetative nitrogen and sulfur to its developing fruit. This study was conducted to determine whether different nitrogen sources affected N and S assimilation and remobilization during pod filling. Well-nodulated plants fed 1.0 mM KNO₃, 0.5 mM urea, or 2.5 mM urea assimilated 0%, 37%, or 114% more N, respectively, and 25%, 46%, or 56% more S, respectively, than did the average non-nodulated control plant fed 5.0 mM KNO₃. Thus, N source during pod filling greatly affected both N and S assimilation. Depending upon N source, plant N concentration during pod filling decreased from 2.96% to between 1.36% and 1.82%. Non-nodulated control plants fed 5.0 mM KNO₃ had the highest residual N at harvest. During the same treatments, plant S concentration decreased from 0.246% to a relatively uniform 0.215%. Thus, during pod filling, vegetative N was seemingly remobilized more efficiently (38–54%) than was S (13%). N source also affected seed yield and seed quality. Non-nodulated control plants fed 5.0 mM KNO₃ produced the lowest yield (21.1 g seeds plant^-1), whereas well nodulated plants fed 1.0 mM KNO₃, 0.5 mM urea, or 2.5 mM urea produced yields of 26.2 g, 31.8 g, and 36.7 g seeds plant^-1, respectively. Non-nodulated plants fed 2.5 mM urea yielded 28.6 g of seeds plant^-1. Seed N concentrations of non-nodulated plants and nodulated plants fed 2.5 mM urea were high, 6.30% and 6.11% N, respectively, whereas their seed S concentrations were low, 0.348% and 0.330% S, respectively. N sources that produced both a relatively high seed yield and seed N concentration (i.e., a relatively high total seed N plant^-1) produced a proportionately smaller increase in total seed sulfur. Consequently, seed quality, as judged solely by seed S concentration, was lowered.     

草酸对土壤胶体与矿物表面酶的吸附及活性影响

采用平衡批处理法,研究了模拟根系分泌物--草酸溶液的浓度、pH对酸性磷酸酶在针铁矿、高岭石及黄棕壤和砖红壤胶体(＜2μm)上的吸附及比活的影响.结果表明,针铁矿对磷酸酶的吸附量受草酸浓度的影响较小,其它供试胶体对蛋白的吸附量随草酸浓度的升高,一般表现为先急剧降低(0～5mmol·L^-1),之后逐渐升高到与对照相当或略低.这与草酸在土壤胶体和矿物表面的配位形态及其对载体表面的电荷改变、溶解有关.草酸体系中,供试胶体对磷酸酶的吸附顺序为针铁矿>黄棕壤＞高岭石＞砖红壤.酶在草酸体系中的最大吸附点位一般出现在蛋白的等电点(IEP)和供试胶体的PZC之间,而酶在草酸体系中被固定到供试胶体上之后,其最适比活点随胶体类型的不同而没有变化或有所高移.     

Effect of silicate on phosphate availability for rice in a P-deficient soil

In a pot experiment the effect of silicate on P availability for rice grown in a P-deficient soil with and without flooding was analyzed. Treatments were designed as follows: C (control: Yakuno soil), SS (sodium silicate application, at 0.47 mg Si g^-1 soil) and SC (sodium carbonate application). In order to separate pH effect from Si effect, SC was adjusted to the same pH as SS.Soil pH of SS and SC increased by 1.0 unit. Shoot dry weight of SC plants, and more so of SS plants, increased under both nonflooded and flooded conditions. P concentrations in the shoots were not increased under either condition of SS and SC. With SS, Si concentration in the shoots significantly increased, Mn concentration significantly decreased, resulting in a higher P/Mn ratio in the shoot, but not with SC. Both SS and SC increased N concentration in the shoots nearly two times compared with control under both conditions.Adsorption experiments showed that neither SS nor SC decreased P adsorption by soil. SS also could not displace the adsorbed P in soil samples which had previously either received P or not.These results suggest that the beneficial effects of silicate on rice growth do not result from increasing P availability in soil. The Si effect may be attributed to decreasing Mn uptake, thus indirectly improving P utilization in the plant.     

Effects of pH and nitrogen on cadmium uptake in potato

This study investigated the effects of pH and nitrogen form and concentration on cadmium (Cd) uptake by potato (Solanum tuberosum L.) grown in hydroponic culture. Potato plants grown in a pH-buffered nutrient solution for 10 d were exposed for 24 h to 25 nM CdCl₂ labelled with ¹⁰⁹Cd. Plants showed a significantly higher Cd uptake and accumulation at pH 6.5 than at pH 4.5 and 5.5. Nitrogen supplied as nitrate (NO₃ ^?) generally resulted in a higher Cd uptake and accumulation than N supplied as ammonium (NH₄ ⁺). This effect was most pronounced at pH 6.5. The N concentration increasing from 6.5 to 26 mM resulted in a decreased Cd influx when either NO₃ ^? or NH₄ ⁺ was used. Cd translocation to the shoot was increased when NO₃ ^? was used as the sole N source. In conclusion, pH had a strong influence on Cd uptake by roots and N form is especially important for Cd translocation within the potato plant.     

Purification and Some Properties of Adenosine (Phosphate) Deaminase from the Liver of the Squid Todarodes pacificus

An enzyme that catalyzed the deamination of adenosine 3′-phenylphosphonate was purified from squid liver to homogeneity as judged by SDS-PAGE. The molecular weight of the enzyme was estimated to be 60,000 by SDS-PAGE and 140,000 by Sephadex G-150 gel filtration. The enzyme deaminated adenosine, 2′-deoxyadenosine, 3′-AMP, and 2′,3′-cyclic AMP, but not adenine, 5′-AMP, 3′,5′-cyclic AMP, ADP, or ATP. The apparent K_m and V_max at pH 4.0 for these substrates were comparable (0.11-0.34mM and 179-295 μmol min^?1 mg^?1, respectively). The enzyme had maximum activity at pH 3.5-4.0 for adenosine 3′-phenylphosphonate, at pH 5.5 for adenosine and 2′-deoxyadenosine, and at pH 4.0 for 2′,3′-cyclic AMP and 3′-AMP when the compounds were at concentration of 0.1 mM. The K_m at 4.0 and 5.5 for each substrate varied, but the Vmax were invariant. These results indicated that the squid enzyme was a novel adenosine (phosphate) deaminase with a unique substrate specificity.     

Die pH-Abhängigkeit der Aufnahme von H2PO 4 - , SO 4 = , Na+ und K+ und ihre gegenseitige Beeinflussung bei Ankistrodesmus braunii

Summary Ion uptake was studied using ³²P, ³⁵S, ²²Na and ⁴²K as tracers in synchronized cells of Ankistrodesmus, which were slightly starved with respect to the ions to be investigated. In the light and in the dark, phosphate uptake is maximal between pH 5.5 and 6.5. Whereas Na⁺ in comparison to K⁺ enhances phosphate uptake in the light (8 to 9-fold) and in the dark, Ca⁺⁺ exerts only a slightly stimulatory effect. The stimulation of phosphate binding by Na⁺ occurs rapidly, even after less than 5 sec of incubation, and also in the presence of an equimolar concentration of K⁺.The pH-dependence of Na⁺-uptake in the light and in the dark is comparable to a dissociation curve: Na⁺-uptake increases with decreasing extracellular H⁺-concentration and is inversely proportional to phosphate uptake in the absence of Na⁺. The light:dark ratio of Na⁺-uptake at pH 8 amounts to 7:1. Mere adsorption of Na⁺ is similarly dependent on the pH. K⁺ strongly competes with Na⁺-uptake, even at pH 8. K⁺-uptake proceeds in a quite different manner from Na⁺-uptake and has an optimum at pH 7.Sulfate is taken up linearly in a biphasic process as a function of time; the pH-optimum lies between pH 7.5 and 8. K⁺ but not Na⁺ slightly enhances sulfate uptake.The Na⁺-enhancement of phosphate uptake can be related neither to a sodium-potassium exchange pump nor to a photosynthesis-dependent ion-exchange reaction.The results suggest that the uptake of phosphate, Na⁺ and K⁺, and the influence of alkali cations on phosphate uptake, but not sulfate uptake, are strongly dependent on fixed charges of the plasmalemma or even of the cell wall. These fixed charges may even prevent an active ion uptake.     

The effect of temperature on the uptake and loss of anions by seedling roots ofZea mays L

Intact maize seedlings were examined for the uptake and leakage of labelled sulphate and phosphate anions affected by temperature. Control plants, grown at 25 °C were compared from the aspects of uptake capacity and leakage with plants incubated in nutrient solutions cooled to 15 °C and 5 °C, respectively. Short time intervals as well as 1–7 d exposure to cooling were used. Already after 1 h exposure at 5 °C and 5 h cooling at 15 °C and at 5 °C, considerable changes were manifested in anion uptake and leakage. The uptake of³²P declined more than that of³⁵S. So, after a 30 min uptake interval the uptake of³⁵S decreased at 15 °C to 49.84% and at 5 °C to 6.05% comparing with the uptake at 25 °C, while the uptake of³²P declined to 28.64% at 15 °C and to 4.45% at 5 °C. The leakage of both anions was the highest at 25 °C in absolute rates, but relatively most of the uptaken³⁵S and³²P was released at 5 °C. Longer exposure to a chilling temperature of 5 °C (1–7 days) resulted in two patterns of sulphate and phosphate uptake.     

Fate of polycyclic aromatic hydrocarbons (PAH) in the rhizosphere and mycorrhizosphere of ryegrass

Polycyclic aromatic hydrocarbons (PAH) can be degraded in the rhizosphere but may also interact with vegetation by accumulation in plant tissues or adsorption on root surface. Previous studies have shown that arbuscular mycorrhizal (AM) fungi contribute to the establishment and maintenance of plants in a PAH contaminated soil. We investigated the fate of PAH in the rhizosphere and mycorrhizosphere including biodegradation, uptake and adsorption. Experiments were conducted with ryegrass inoculated or not with Glomus mosseae P2 (BEG 69) and cultivated in pots filled with soil spiked with 5 g kg⁻¹ of anthracene or with 1 g kg⁻¹ of a mixture of 8 PAH in a growth chamber. PAH were extracted from root surfaces, root and shoot tissue and rhizosphere soil and were analysed by GC-MS. In both experiments, 0.006 – 0.11‰ of the initial extractable PAH concentration were adsorbed to roots, 0.003 – 0.16‰ were found in root tissue, 0.001‰ in shoot tissue and 36 – 66% were dissipated, suggesting that the major part of PAH dissipation in rhizosphere soil was due to biodegradation or biotransformation. With mycorrhizal plants, anthracene and PAH were less adsorbed to roots and shoot tissue concentrations were lower than with non mycorrhizal plants, which could contribute to explain the beneficial effect of AM fungi on plant survival in PAH contaminated soils. This revised version was published online in June 2006 with corrections to the Cover Date.     

Effect of Phosphorus Deficiency on Phosphatase Activity of Cell Walls from Roots of Subterranean Clover

Clover (Trifolium subterraneum L. cv. Mt. Barker) was grownin solution culture with adequate (+P) or no phosphate (–P).Cell walls were extracted from roots in such a way that theywere uncontaminated by other cellular materials. Phosphataseactivity was assayed using p-nitro-phenylphosphate (NPP). Phosphatasebound to cell walls had a pH optimum between 5.0 and 6.0, irrespectiveof the P supply to the plants. Activity of phosphatase boundto cell walls increased with electrolyte concentration of theassay medium at pH 6.5 but not at pH 5.5. This increase in activitywas probably due to a higher degree of ionization of the cellwall at pH 6.5 than at pH 5.5, and to effects of high ionicstrength in decreasing the mutual repulsion of negatively chargedNPP from negative charges on the cell walls. Cell wall-boundphosphatase did not exhibit Michaelis-Menten kinetics: the concentrationof NPP at which activity was half the maximum rate (S_0.5) was0.7 mM for cell walls extracted from roots of both +P and –Pplants. Up to 30% of the phosphatase activity bound to cellwalls could be removed using buffer solutions of high pH andhigh ionic strength which contained Triton X100. Both soluble and cell wall-bound phosphatase(s) of roots increasedin activity with P deficiency. The phosphatase activity of cellwalls increased 1.5 fold as the P concentration in the rootsfell from 0.4–0.2% dry weight. Experiments with sterileroots of clover showed that increases in cell wall-bound phosphataseactivity associated with P deficiency were not due to microbialcontamination. It is argued that phosphatase(s) in cell wallsof roots could make a substantial contribution to the P nutritionof clover in soils deficient in inorganic phosphate by hydrolysingorganic phosphate compounds in the soil. Key words: Phosphatase, Clover, Roots, Phosphorus deficiency, Cell walls