Interactive effects of mycorrhization and elevated atmospheric CO2 on sulphur nutrition of young pedunculate oak (Quercus robur L.) trees

Pedunculate oak (Quercus robur L.) was germinated and grown at ambient CO₂ concentration and 650 μmol mol^?1 CO₂ in the presence and absence of the ectomycorrhizal fungus Laccaria laccata for a total of 22 weeks under nonlimiting nutrient conditions. Sulphate uptake, xylem loading and exudation were analysed in excised roots. Despite a relatively high affinity for sulphate (K_M= 1.6 mmol m^?3), the rates of sulphate uptake by excised lateral roots of mycorrhizal oak trees were low as compared to herbaceous plants. Rates of sulphate uptake were similar in mycorrhizal and non-mycorrhizal roots and were not affected by growth of the trees at elevated CO₂. However, the total uptake of sulphate per plant was enhanced by elevated CO₂ and further enhanced by elevated CO₂ and mycorrhization. Sulphate uptake seemed to be closely correlated with biomass accumulation under the conditions applied. The percentage of the sulphate taken up by mycorrhizal oak roots that was loaded into the xylem was an order of magnitude lower than previously observed for herbaceous plants. The rate of xylem loading was enhanced by mycorrhization and, in roots of mycorrhizal trees only, by growth at elevated CO₂. On a whole-plant basis this increase in xylem loading could only partially be explained by the increased growth of the trees. Elevated CO₂ and mycorrhization appeared to increase greatly the sulphate supply of the shoot at the level of xylem loading. For all treatments, calculated rates of sulphate exudation were significantly lower than the corresponding rates of xylem loading of sulphate. Radiolabelled sulphate loaded into the xylem therefore seems to be readily diluted by unlabelled sulphate during xylem transport. Allocation of reduced sulphur from oak leaves was studied by flap-feeding radiolabelled GSH to mature oak leaves. The rate of export of radioactivity from the fed leaves was 4–5 times higher in mycorrhizal oak trees grown at elevated CO₂ than in those grown at ambient CO₂. Export of radiolabel proceeded almost exclusively in a basipetal direction to the roots. From these experiments it can be concluded that, in mycorrhizal oak trees grown at elevated CO₂, the transport of sulphate to the shoot is increased at the level of xylem loading to enable increased sulphate reduction in the leaves. Increased sulphate reduction seems to be required for the enhanced allocation of reduced sulphur to the roots which is observed in trees grown at elevated CO₂. These changes in sulphate and reduced sulphur allocation may be a prerequisite for the positive effect of elevated CO₂ on growth of oak trees previously observed.     

Sulphate transport in the cyanobacterium Synechococcus R-2 (Anacystis nidulans, S. leopoliensis) PCC 7942

Synechococcus R-2 (PCC 1942) actively accumulates sulphate in the light and dark. Intracellular sulphate was 1.35 ± 0.23 mol m^?3 (light) and 0.894 ± 0.152 mol m^?3 (dark) under control conditions (BG-11 media: pH_o, 7.5; [SO₄^2?]_o, 0.304 mol m^?3). The sulphate transporter is different from that found in higher plants: it appears to be an ATP-driven pump transporting one SO₄^2?/ATP [ΔμSO₄^2?_i,o=+ 27.7 ± 0.24 kJ mol^?1 (light) and + 24 ± 0.34 kj mol^?1 (dark)]. The rate of metabolism of SO₄^2?at pH_o, 7.5 was 150 ± 28 pmol m^?2 s^?1 (n = 185) in the light but only 12.8 ± 3.6 pmol m^?2 s^?1 (n = 61) in the dark. Light-driven sulphate uptake is partially inhibited by DCMU and chloramphenicol. Sulphate uptake is not linked to potassium, proton, sodium or chloride transport. The alga has a constitutive over-capacity for sulphate uptake [light (n= 105): K_m= 0.3 ± 0.1 mmol m^?3, V_max, = 1.8 ± 0.6 nmol m^?2 s^?1; dark (n= 56): K_m= 1.4 ± 0.4 mmol m^?3, V_max= 41 ± 22 pmol m^?2 s^?1]. Sulphite (SO₃^2?) was a competitive inhibitor of sulphate uptake. Selenate (SeO₄^2?) was an uncompetitive inhibitor.     

Phosphate transport in fertilized sea urchin eggs. I. Kinetic aspects

Properties of the fully developed phosphate transport system in the fertilized egg of the sea urchin, Strongylocentrotus purpuratus, were investigated. The rates of phosphate transport at concentrations of external phosphate of 1 to 44 μM, both in the absence and in the presence of 100 μM arsenate, exhibit typical saturation kinetics. At sea water concentrations of 2 μM phosphate, the rate of uptake is about 2 × 10^?9 μm/egg/minute at 15°C. Arsenate is a competitive inhibitor of phosphate transport, fully and immediately reversible in its effects, yielding K_i values ranging from 10.5 to 14.1 × 10^?6 M in comparison to the corresponding apparent K_M (Michaelis-Menten) constants for phosphate of 5.6 to 7.5 × 10^?6 M (pH 8.0, 15°C). The rate of arsenate uptake in a phosphate deficient medium amounts to 2.8 to 2.9 × 10^?10 μm arsenate/egg/minute at an arsenate concentration of 2.9 to 10.2 μM arsenate (HAsO₄^??), which is 9.5 and 5.6% of the rate of phosphate uptake at corresponding phosphate concentrations. Arsenate has essentially the same developmental effects at initial concentrations of 5–10 μM and 100 μM arsenate, namely no observable effects for exposure periods of 7.5 hours, although longer periods result in blockage of development at the early blastula stage. Outward flux of phosphate ions cannot be demonstrated by washing prelabelled eggs with sea water containing low or high concentrations of phosphate, even when phosphorylation has been blocked by exposing the eggs to a metabolic inhibitor. Phosphate uptake rates measured in the pH range from 5.0 to 10.0 reveal a sharp optimum at pH 8.8–8.9. Reference to the apparent pK' values of the phosphoric acid system indicate that the entering species is the HPO₄^?? ion. The effects on rates of phosphate uptake of exposure to sea water at pH values between 7 and 10 for 30 minute periods are fully reversible, but at lower pH values, reversal is delayed, and is only partial. Sodium molybdate (0.01 M), sodium pyrophosphate (1.5 × 10^?4 M), and adenosine triphosphate (1–5 × 10^?4 M) for exposure periods ranging from 40 to 180 minutes did not significantly affect phosphate uptake. Omission of Ca⁺⁺ ion from artificial sea water is without effect on phosphate uptake but the absence of both Ca⁺⁺ and Mg⁺⁺ results in profound and irreversible depression of both phosphate uptake and development. The data of this and the following paper are consistent with the conclusion that the transport of phosphate involves a surface located carrier. The apparent secondary and tertiary ionization constants of phosphoric acid in sea water (ionic strength = 0.6885) were measured, resulting in a value for pK′₂ = 6.14 and for pK′₃ = 10.99, at 15°C and phosphate at infinite dilution.     

Sulphate/proton cotransport in plasma-membrane vesicles isolated from roots of Brassica napus L.: increased transport in membranes isolated from sulphur-starved plants

The characteristics of sulphate uptake into right-side-out plasma-membrane vesicles isolated from roots of Brassica napus L., Metzger, cv. Drakkar, and purified by aqueous polymer two-phase partitioning, were investigated. Sulphate uptake into the vesicles was driven by an artificially imposed pH gradient (acid outside), and could be observed for 5–10 min before a plateau was reached and no further net uptake occurred. The uptake was partially inhibited in the presence of depolarizing agents and little uptake was observed in the absence of an imposed pH gradient. Uptake was strongly pH-dependent, being greatest at more acidic pH. After imposition of a pH gradient, the capacity for uptake decreased slowly (t1/2>10 min). The uptake had a high-affinity component which was strongly dependent on the external proton concentration (K _m=10μM at pH 5.0, 64 μM at pH 6.5). The K _m for protons varied from 0.4–1.9 μM as the sulphate concentration was reduced from 33 to 1 μM. A low-affinity component was observed which could be resolved at low temperatures (0 °C). Microsomal membranes that partitioned into the lower phase of the two-phase system gave no indication of high-affinity sulphate transport. Sulphate uptake into plasma-membrane vesicles isolated from sulphur-starved plant material was approximately twofold greater than that observed in those isolated from sulphate-fed plant material. Isolated vesicles therefore mirror the well-known in-vivo response of roots, indicating an increase in the number of transporters to be, at least in part, the underlying cause of derepression.     

Sulphate uptake and metabolism in the chrysomonad,Monochrysis lutheri

The intracellular concentration of inorganic ³⁵SO₄ in Monochrysis lutheri cells exposed to 0.513 mM Na₂ ³⁵SO₄ for up to 6-hr remained constant at about 0.038 mM. The exchange rate of this ³⁵SO₄ with the external unlabelled sulphate was negligible compared to the rate of influx across the plasmalemma (0.032 μmoles/g cells/hr). The flux of free ³⁵SO₄ to organic ³⁵S was 0.029 μmoles/g cells/hr. Assuming an internal electrical potential in the cells of-70 mV, this intracellular concentration of inorganic ³⁵SO₄ was well in excess of that obtainable by passive diffusion as calculated from the Nernst equation. These results indicate that sulphate is accumulated by an active mechanism rather than by facilitated diffusion. Sulphate uptake appears to occur via a carrier-mediated membrane transport system which conforms to Michaelis-Menten type saturation kinetics with a K _m of 3.2×10^-5 M and a V _max of 7.9×10^-5 μmoles sulphate/hr/10⁵ cells. Uptake was dependent on a source of energy since the metabolic inhibitor CCCP almost completely inhibited uptake under both light and dark conditions and DCMU caused a 50% decrease in uptake under light conditions. Under dark conditions, uptake remained at about 80% of that observed under light conditions and was little affected by DCMU, indicating that the energy for uptake could be supplied by either photosynthesis or respiration. A charge and size recognition site in the cell is implied by the finding that sulphate uptake was inhibited by chromate and selenate but not by tungstate, molybdate, nitrate or phosphate. Chromate did not inhibit photosynthesis. Cysteine and methionine added to the culture medium were apparently capable of exerting inhibition of sulphate uptake in both unstarved and sulphate-starved cells. Cycloheximide slightly inhibited sulphate uptake over an 8-hr period indicating, either a slow rate of entry of the inhibitor into the cells or a slow turnover of the proteins(s) associated with sulphate transport.     

High-Affinity Uptake of [3H]Norepinephrine by Primary Astrocyte Cultures and Its Inhibition by Tricyclic Antidepressants

Abstract: Primary astrocyte cultures from neonatal rat brains show uptake of [³H]norepinephrine ([³H]NE). This uptake has a high-affinity component with an apparent K_m of approximately 3 × 10^?7 M. At 10^?7 M [³H]NE both the initial rate of uptake and steady-state content of [³H]NE is inhibited by up to 95% by omission of external Na⁺. The Na⁺-dependent component of this uptake is totally inhibited by the tricyclic antidepressants desipramine (DMI) and amitryptyline with IC₅₀ values of 2 × 10^?9 and 4 × 10^?8 M, respectively. Inhibition of [³H]NE uptake by DMI shows competitive kinetics. These characteristics are essentially identical to those found for high-affinity uptake of NE in total membrane or synaptosome fractions from rodent brains and suggests that such uptake in neural tissue is not exclusively neuronal.     

METHYLAMINE UPTAKE IN THE GREEN ALGA CHLORELLA PYRENOIDOSA1

Methylamine uptake in nitrogen-starved Chlorella pyrenoidosa Beij. follows Michaelis-Menten kinetics: maximum uptake is about 1.6 nmol μl^?1· cells · min^?1, half-saturation occurs at 4 μM methylamine, and the slope in the range where uptake is proportional to concentration is 0.4 nmol μl^?1· min^?1·μM^?1. In cells grown in the presence of a non-limiting nitrogen concentration, methylamine uptake is directly proportional to concentration up to at least 0.5 mM, and the slope is 1/500 that for starved cells. Similar uptake kinetics have been reported for Penicillium chrysogenum and attributed to an inducible “ammonium permease.” Apparently, a similar permease occurs in algae.     

Adaptation to chilling: photosynthetic characteristics of the cultivated tomato and a high altitude wild species

Abstract When tomato plants of the high-altitude species Lycopersicon hirsutum and of the cultivated Lycopersicon esculentum were grown at 24/18°C (day/night), the effects of temperature, photon flux density, and intercellular CO₂ concentration up to about 600 μl l^?1 on net CO₂ uptake were similar in the two species. Acclimation of these plants at 12/6°C (day/night) resulted, after 4 d or longer, in a similar downward shift of about 5°C in the optimum temperature for CO₂ uptake. However, in comparison with the cultivated species, the high-altitude plants achieved a higher rate of CO₂ uptake at saturating concentrations of intercellular CO₂, maintained a higher level of saturating-light CO₂ uptake rate at 10°C after exposure to chilling stress (10°C and photon flux density of 400 μmol m^?2s^?1 d and 5°C night) for 7–18 d, and displayed a better capacity for rapid recovery after prolonged stress. The greater capacity for CO₂ uptake observed in the high-altitude species during and after exposure to chilling stress was also reflected in its higher growth rate under those conditions compared with plants of L. esculentum. These advantages of the high-altitude species may partly explain its ability to survive and complete its life cycle under the environmental conditions prevailing in its natural habitat.     

Dimeric chlorite dismutase from the nitrogen‐fixing cyanobacterium Cyanothece sp. PCC7425

It is demonstrated that cyanobacteria (both azotrophic and non‐azotrophic) contain heme b oxidoreductases that can convert chlorite to chloride and molecular oxygen (incorrectly denominated chlorite ‘dismutase’, Cld). Beside the water‐splitting manganese complex of photosystem II, this metalloenzyme is the second known enzyme that catalyses the formation of a covalent oxygen–oxygen bond. All cyanobacterial Clds have a truncated N‐terminus and are dimeric (i.e. clade 2) proteins. As model protein, Cld from Cyanothece sp. PCC7425 (CCld) was recombinantly produced in Escherichia coli and shown to efficiently degrade chlorite with an activity optimum at pH 5.0 [k_cat 1144 ± 23.8 s^?1, K_M 162 ± 10.0 μM, catalytic efficiency (7.1 ± 0.6) × 10⁶ M^?1 s^?1]. The resting ferric high‐spin axially symmetric heme enzyme has a standard reduction potential of the Fe(III)/Fe(II) couple of ?126 ± 1.9 mV at pH 7.0. Cyanide mediates the formation of a low‐spin complex with k_on = (1.6 ± 0.1) × 10⁵ M^?1 s^?1 and k_off = 1.4 ± 2.9 s^?1 (K_D ～ 8.6 μM). Both, thermal and chemical unfolding follows a non‐two‐state unfolding pathway with the first transition being related to the release of the prosthetic group. The obtained data are discussed with respect to known structure–function relationships of Clds. We ask for the physiological substrate and putative function of these O₂‐producing proteins in (nitrogen‐fixing) cyanobacteria.     

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%.     

Modulation of sprouting in organ culture after axotomy of an identified molluscan neuron

We examined a variety of factors that might modulate the initiation of neurite outgrowth in an attempt to identify means by which its initiation might be accelerated. We examined this initiatio from an identified molluscan neuron, Helisoma trivolvis buccal neuron B5 after axotomy, and determined whether the site of injury, temperature, ion channel blockers, pH, the second messenger cAMP, and protein synthesis affect the initiation of neurite outgrowth. Neurite outgrowth was assayed from axotomized neurons by filling the neurons intracellularly with Lucifer Yellow and examining the percentage of axons that extended (sprouted) new process after 9 or 24 h in organ culture. About one-third (31%) of axotomized neurons sprouted from the site of injury after 9 h (n = 22), and 88% (n = 20) sprouted after 24 h in saline at 22°–24°C when the injury was located 800 μm from the soma. Elevating the temperature to 32°C or moving the lesion site to 400 or 1500 μm from the soma did not significantly alter the incidence of sprouting. Blocking sodium channels with tetrodotoxin [TTX (2 × 10^?5 M)] did not significantly reduce the incidence of sprouting, whereas the sodium channel agonist, veratridine (10^?5 M) did. The calcium channel blocker lanthanum (10^?6–10^?4 M), stimulated neurite outgrowth; however, the organic calcium channel blocker verapamil (10^?3–10^?5 M), and the calcium ionophore A23187 (10^?5 M), had no effect on sprouting. Exposure of neurons to the potassium channel blocker tetraethylammonium [TEA (20 mM)], elevation of intracellular pH with NH₄Cl (5 mM), or treatment with the adenylate cyclase activator forskolin (10^?5 M) reduced the incidence of sprouting, whereas dideoxy-forskolin (10^?5 M) had no effect. Inhibition of protein synthesis with anisomycin (2 × 10^?4 to 2 × 10^?6 M) did not significantly suppress sprouting 24 h after axotomy. Both d and l isomers of glutamate (300 μM) stimulated sprouting. The present results suggest that the initiation of sprouting is regulated locally at or near the site of injury, and that blocking specific ion channels may either inhibit or enhance the initiation of neurite outgrowth.     

Properties of glutamate dehydrogenase from Beta vulgaris

Glutamate-NAD oxidoreductase, E.C. 1.4.1.3 (GDH), from seedlings of Beta vulgaris cv. Rota, Jahnsch Peragis Comp., was enzymatically characterized. This enzyme with molecular weight of 2.6 × 10⁵ has a pH optimum of around 8 for animation of α-KGA and around 9.5 for the desamination of glutamate. The apparent K_m for α-KGA is 6.7 × 10^?4M, for NH₃ 2.5 × 10^?3M, for NADH 3.2 × 10^?5M and for NAADPH 5.5 × 10^?4M. NAD¹ inhibits the reaction non-competitively when NADPH serves as substrate. The apparent K¹ is 4.5 × 10^?4M. The data are discussed on relation to the properties of GDH from other plant sources.     

Sulphate reduction in the hypolimnion and sediments of Lake Kinneret, Israel

1. Lake Kinneret is a warm (13–30°C) monomictic lake. Between January and June a heavy annual bloom of the dinoflagellate Peridinium gatunense dominates phytoplankton biomass (250 g m^?2). At the beginning of the summer, degradation and decomposition of the Peridinium biomass occurs, serving as a trigger for intense sulphate reduction in the hypolimnion and sediments. 2. The rates of sulphate reduction in the sediments varied seasonally from 12 to 1700 nmol SO₄.^?2 reduced cm^?3 day^?1 in December and July, respectively. The availability of organic matter and sulphate is high in June after the crash of the Peridinium bloom and the beginning of stratification and is lowest in December before overturn. 3. Sulphate concentrations in the hypolimnion range between 0.52 mM and 0.20 mM during mixing (January-April) and before overturn (December), respectively. The depletion in sulphate in the hypolimnion is stoichiometrically correlated to the increase in sulphide. The lake is not depleted of sulphate at any time, so the sulphate reduction process in Lake Kinneret is not limited by sulphate concentrations except in the sediments just before overturn.     

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.     

Glycolate metabolism in cyanobacteria. IV. Uptake, growth and metabolic pathways

Anacystis nidulans (UTEX 625) and Anabaena cylindrical (CCAP 1403/2a) incorporated minor quantities of [¹⁴C]-glycolate via diffusion, whereas Plectonema boryanum (PCC 73110) and Nostoc 268 rapidly incorporated [¹⁴C]-glycolate. A carrier mediated uptake across the membrane is suggested for the two latter strains. In these strains the initial [¹⁴C]-glycolate incorporation (>30 s) was inhibited by the uncoupler m-chlorophenylhydrazone and the F₀F₁-ATPase inhibitor N,N′-dicyelohcxylearbodiimide (DCCD) but was not affected by inhibitors of glycolate metabolism: 2-pvridyl-hydroxymethanesulfonic acid (HPMS), glycidate, aminooxyacetic acid and aminoacetoniirile. The incorporation rate was about 0.5 and 40 umol (ma chl a)^?1 h^?1 at 17 μM and 5 mM glycolate, respectively, Anacystis nidulans did not grow on gtycolate. whereas Anabaena cylindrical to some extent did which suggests an inducible glycolate uptake system in this strain. Anahaena 7120 and Nostoc 268 grew photoheterotrophically on glycolate. The reduced [¹⁴C]-glvcolale uptake by Anabaena 7120 in the presence of glycidate. aminooxyaeetic acid and aminoacetonitrile indicates that in the light, a large part of the [¹⁴C]-glycolate incorporated was metabolized via glycine to serine. The net uptake of [¹⁴C]-glycolate and the effect of different inhibitors was dependent on the source of nitrogen used (for growth and the nitrogen status during the assay. In cells cultivated in N-free medium (nitrogen-fixing cells) a larger part of the [¹⁴C]-glycolate seemed to be metabolized via glycine to serine compared to that in cells cultivated in presence of NH₄Cl (nonnitrogen-fixing cells). The capacity to incorporate [¹⁴C]-glyeolate by non-nitrouen-fixing cells was enhanced in presence of NH₄CI.     

Sulphate diffusion and incorporation into human articular cartilage

The permeability coefficients of sulphate ion in post-mortem human articular cartilage were found to be the same whether cells were alive or dead; thus diffusion of solutes is not via active transport. From the diffusion coefficient and the thickness of cartilage, the minimum time of incubation necessary to obtain meaningful results on sulphate uptake and incorporation, could be calculated.The rate of ³⁵S-labelled sulphate incorporation was linear up to 8 h. In Eagle's medium, the mean rates of incorporation, in mmoles/gram of wet tissue per h were 2 · 10^?6 for the femoral head and 3.3 · 10^?6 for the femoral condyle. The faster turnover rate in the condyle correlates with a lower glycosaminoglycan content.Sulphate uptake was found to vary directly with the inorganic sulphate content. Since the latter by Donnan equilibrium, is in inverse ratio to the glycosaminoglycan content, this would explain why sulphate uptake was found to be lower where the glycosaminoglycan content was higher.The half-life of glycosaminoglycans was estimated at 200–300 days i.e. much higher than previously suggested.Zonal variations in uptake were studied both in normal and fibrillated tissue; the latter has a low rate of incorporation, throughout its depth, compared to healthy cartilage.     

Constitutive and inducible aspects of nitrate-nitrogen uptake by Chlamydomonas reinhardtii

Similarly to higher plant root systems, Chlamydomonas reinhardtii Dangeard (UTEX 90) cells exhibited biphasic NO₃^? uptake kinetics. The uptake pattern was similar in cells cultured in 10 mM NO₃^? (NO₃^?-grown), 0.25 mM NO₃^? (N-limited) or 10 mM NO₃^? followed by an 18-h period of N-deprivation (N-starved). In all cell types there was an apparent phase transition in uptake at 1.1 mM NO₃^?, although there were variations in the uptake V_max of both isotherms. The rate of uptake via isotherm 0 ([NO₃^?]<1.1 mM) in N-limited cells was higher than that of either NO₃^?-grown or N-starved cells. In contrast, NO₃^?-grown and N-limited cells exhibited comparable V_max values when supplied with 1.1 to 1.8 mM NO₃^? (isotherm 1). When supplied with 1.6 mM NO₃^?, both N-limited and N-starved cells exhibited enhanced linear uptake after 60 min of incubation. We ascribed this to an induction phenomenon. This trend was not observed when NO₃^?-grown cells were supplied with 1.6 mM NO₃^?, or when N-limited and N-starved cells were supplied with 0.6 mM NO₃^?. The ‘inducible’ aspect of uptake by N-limited cells was blocked by cycloheximide (10 mg l^?1), but not by actinomycin D (5 mg l^?1), thus indicating the involvement of a translational or post-translational event. To investigate this phenomenon further, we analysed the cell proteins of N-limited cells supplied with either 0.6 or 1.6 mM NO₃^? for 90 min, using two-dimensional gel electrophoresis. Comparison of protein profiles enabled the identification of a single cell membrane-associated polypeptide (21 kDa, pI ca 5.5) and ten soluble fraction polypeptides (17–73 kDa, pI ca 5.0 to 7.1) unique to the high NO₃^? treatment. We propose that the ‘inducible’ portion of NO₃^? uptake may provide the means by which C. reinhardtii cells regulate uptake in accordance with assimilatory capacity.     

Studies on sulphur-selenium antagonism in blue-green algae

Summary Anacystis nidulans and Anabaena variabilis contain sufficient sulphur reserves to enable them to perform only one round of growth cycle in the non-sulphur growth medium. Sulphate, sulphite, l-methionine and d-methionine, each can act as a suitable sulphur source, but they differ in respect of their growth promoting action; sulphate uptake seems to be a light driven phenomenon and the sulphate metabolizing enzymes are inducible in nature. Methionine appears to act as a repressor of sulphate-metabolizing enzymes.     

Multiphasic uptake of potassium by barley roots of low and high potassium content: Separate sites for uptake and transitions

In the range 10^?6M - 5 × 10^?2M uptake of K⁺ in excised roots of barley (Hordeum vulgare L. cv. Herta) with low and high K content could in both cases be represented by an isotherm with four phases. Uptake, especially in the range of the lower phases, was reduced in high K roots through decreases in V_max and increases in K_m. Similar data for other plants are also shown to be consistent with multiphasic kinetics. The concentrations at which transitions occurred were not affected by the K status, indicating the existence of separate uptake and transition sites. Uptake was markedly reduced in the presence of 10^?5M 2,4-dinitrophenol, especially at low K⁺ concentrations, but the isotherms remained multiphasic. This contraindicates major contributions from a non-carrier-mediated, passive flux. A tentative hypothesis for multiphasic ion uptake envisions a structure which changes conformation as a result of all-or-none changes in a separate transition site. The structure is “tight” at low external ion concentrations (low V_max. low K_m. active uptake, allosteric regulation) and “loose” at high concentrations (high V_max- high K_m- facilitated diffusion, no regulation).     

Purification and characterization of laccase secreted by Phellinus linteus MTCC-1175 and its role in the selective oxidation of aromatic methyl group

A laccase from the culture filtrate of Phellinus linteus MTCC-1175 has been purified to homogeneity. The method involved concentration of the culture filtrate by ammonium sulphate precipitation and an anion exchange chromatography on DEAE-cellulose. The SDS-PAGE and native-PAGE gave single protein band indicating that the enzyme preparation was pure. The molecular mass of the enzyme determined from SDS-PAGE analysis was 70 kDa. Using 2.6-dimethoxyphenol, 2.2′[azino-bis-(3-ethylbonzthiazoline-6-sulphonic acid) diammonium salt] (ABTS) and 4-hydroxy-3,5-dimethoxybenzaldehyde azine as the substrates, the K _m, k _cat and k _cat/K _m values of the laccase were found to be 160 μM, 6.85 s^?1, 4.28 × 10⁴ M^?1 s^?1, 42 μM, 6.85 s^?1, 16.3 × 10⁴ M^?1 s^?1 and 92 μM, 6.85 s^?1, 7.44 × 10⁴ M^?1 s^?1, respectively. The pH and the temperature optima of the P. linteus MTCC-1175 laccase were 5.0 and 45°C, respectively. The activation energy for thermal denaturation of the enzyme was 38.20 kJ/mole/K. The enzyme was the most stable at pH 5.0 after 1 h reaction. In the presence of ABTS as the mediator, the enzyme transformed toluene, 3-nitrotoluene and 4-chlorotoluene to benzaldehyde, 3-nitrobenzaldehyde and 4-chlorobenzaldehyde, respectively.