Concentrations Of Sodium Phosphate Research Articles

Fosfato de sódio e cloreto de potássio na micropropagação de videira e amoreira-preta

This work aimed to test different concentrations of sodium phosphate (NaH 2 PO 4 .H 2 O) and potassium chloride (KCl) taken from the White medium and added to the Knudson culture medium, in the in vitro multiplication of grapevine rootstocks and blackberry. The culture medium was constituted of Knudson salts, added of 25 g L -1 of sucrose and 6 g L -1 of agar, and the adjusted pH to 5.8 before the sterilization at 121oC and 1 atm for 20 minutes. The treatments consisted of concentrations of NaH 2 PO 4 .H 2 O(0, 125, 250, 500 and 1000 mg L -1 ) and of KCl (0, 125, 250, 500 and 1000 mg L -1 ), in all possible combinations, and of grapevine rootstocks ‘R110’ and blackberry cv. Cherokee. Nodal segments originated from in vitro pre-established plants were excised and introduced in test tubes containing 15 mL of culture medium. Subsequently, the tubes were transferred into a growth room at 25 ± 2oC, irradiance of 32 umol m -2 s -1 and photoperiod of 16 hours. The experiment design was completely randomized, with 4 repetitions and 12 plants each. The experiment was evaluated after 60 days of in vitro cultivation. The largest length of the aerial part and fresh weight of the callus were verified in the Knudson culture medium, in the absence of potassium chloride. The addition of potassium chloride in the Knudson culture medium did not influence length increase of the aerial part in both fruit trees; neither did it influence the increase of fresh mass of callus of grapevine rootstocks ‘R110’. It is not necessary to add sodium phosphate to Knudson in order to obtain larger fresh mass in the aerial part of both fruit trees.

Glutamine Deamidation Destabilizes Human γD-Crystallin and Lowers the Kinetic Barrier to Unfolding

Human eye lens transparency requires life long stability and solubility of the crystallin proteins. Aged crystallins have high levels of covalent damage, including glutamine deamidation. Human gammaD-crystallin (HgammaD-Crys) is a two-domain beta-sheet protein of the lens nucleus. The two domains interact through interdomain side chain contacts, including Gln-54 and Gln-143, which are critical for stability and folding of the N-terminal domain of HgammaD-Crys. To test the effects of interface deamidation on stability and folding, single and double glutamine to glutamate substitutions were constructed. Equilibrium unfolding/refolding experiments of the proteins were performed in guanidine hydrochloride at pH 7.0, 37 degrees C, or urea at pH 3.0, 20 degrees C. Compared with wild type, the deamidation mutants were destabilized at pH 7.0. The proteins populated a partially unfolded intermediate that likely had a structured C-terminal domain and unstructured N-terminal domain. However, at pH 3.0, equilibrium unfolding transitions of wild type and the deamidation mutants were indistinguishable. In contrast, the double alanine mutant Q54A/Q143A was destabilized at both pH 7.0 and 3.0. Thermal stabilities of the deamidation mutants were also reduced at pH 7.0. Similarly, the deamidation mutants lowered the kinetic barrier to unfolding of the N-terminal domain. These data indicate that interface deamidation decreases the thermodynamic stability of HgammaD-Crys and lowers the kinetic barrier to unfolding due to introduction of a negative charge into the domain interface. Such effects may be significant for cataract formation by inducing protein aggregation or insolubility.

Examination of Key Intermediates in the Catalytic Cycle of Aspartate-β-semialdehyde Dehydrogenase from a Gram-positive Infectious Bacteria

Aspartate-beta-semialdehyde dehydrogenase (ASADH) catalyzes a critical branch point transformation in amino acid bio-synthesis. The products of the aspartate pathway are essential in microorganisms, and this entire pathway is absent in mammals, making this enzyme an attractive target for antibiotic development. The first structure of an ASADH from a Gram-positive bacterium, Streptococcus pneumoniae, has now been determined. The overall structure of the apoenzyme has a similar fold to those of the Gram-negative and archaeal ASADHs but contains some interesting structural variations that can be exploited for inhibitor design. Binding of the coenzyme NADP, as well as a truncated nucleotide analogue, into an alternative conformation from that observed in Gram-negative ASADHs causes an enzyme domain closure that precedes catalysis. The covalent acyl-enzyme intermediate was trapped by soaking the substrate into crystals of the coenzyme complex, and the structure of this elusive intermediate provides detailed insights into the catalytic mechanism.

On the Origin of the Thermostabilization of Proteins Induced by Sodium Phosphate

The B1 domain of protein L shows a linear rise in thermostability with increasing concentrations of sodium phosphate. Equal behavior is observed for a set of mutant proteins where surface lysines are mutated to noncharged residues, but the mutant's thermostabilities show different sensitivities to phosphate, encoded in the varying slopes observed (mi). The melting temperature in the absence of the cosolute also correlates linearly with mi. The stabilizing effect of the phosphate ion reaches a saturation point, which has been experimentally determined for protein L (1610 mM phosphate). These results indicate that the phosphate-induced stabilization is an inherent property of the protein, encoded in the amino acid sequence. Changes in stability upon mutation are attributed to a redistribution of the overall network of solvated surface charges. Stabilization by phosphate is understood in terms of interactions with the protein surface, reducing the unfavorable contacts between like charges, maximizing the number of accessible conformations of the surface-charged side chains, and optimizing solvation.

Application of Design-of-Experiments Procedures to Optimize Efficiently Pretreatment of Lipase for Use in a Nonaqueous Reaction

A variety of different pretreatments can improve the performance of enzymes in nonpolar reaction media. These pretreatments have primarily been studied in isolation; however, interactions between some pairs of pretreatments are known to exist. The presence of these interactions complicates the design of an optimum multifactor pretreatment. Modern design-of-experiments techniques allow the simultaneous optimization of two or more variables. To improve the performance of lipase in a model reaction, we used a technique called the method of steepest ascent to optimize three variables simultaneously: pretreatment pH and sodium phosphate concentration, and the concentration of acetic acid (one of the reactants) in the reaction mixture. In only 26 experimental runs, this optimization process determined a combination of variable settings that yielded a reaction product approx 180 times faster than achieved with untreated enzyme. Evidence is presented to demonstrate that locating this optimum with single-factor experiments would be inefficient. This article demonstrates the efficiency of the method of steepest ascent particularly for evaluation of enzymatic reaction conditions exhibiting significant interactions.

Factors Affecting Autocatalysis of Botulinum A Neurotoxin Light Chain*

The light chain of botulinum neurotoxin serotype A undergoes autocatalytic fragmentation into two major peptides during purification and storage (Ahmed S. A. et al. 2001, J. Protein Chem. 20:221-231) by both intermolecular and intramolecular mechanisms (Ahmed S. A. et al. 2003, Biochemistry 42:12539 12549). In this study, we investigated the effects of buffers and salts on this autocatalytic reaction in the presence and absence of zinc chloride. In the presence of zinc chloride, the fragmentation reaction was enhanced in each of acetate, MES, HEPES and phosphate buffers with maximum occurring in acetate when compared to those in the absence of zinc chloride. Adding sodium chloride in phosphate buffer in the presence of zinc chloride increased the extent of proteolysis. Irrespective of the presence of zinc chloride, adding sodium chloride or potassium chloride in phosphate buffer elicited an additional proteolytic reaction. Higher concentrations of sodium phosphate buffer enhanced the autocatalytic reaction in the absence of zinc chloride. In contrast, in the presence of zinc chloride, higher concentrations of sodium phosphate decreased the autocatalytic reaction. Optimum pH of autocatalysis was not affected significantly by the absence or presence of zinc chloride. Like zinc chloride, other chlorides of divalent metals, such as magnesium, cobalt, iron and calcium also enhanced the autocatalytic reaction. Polyols such as ethylene glycol protected the light chain from fragmentation. Exposure of light chain to UV radiation led to enhanced fragmentation. In order to avoid fragmentation, the protein should be stored frozen in a low concentration buffer of neutral or higher pH devoid of any metal. Our results provide a choice of buffers and salts for isolation, purification and storage of intact botulinum neurotoxin serotype A light chain.

Partitioning of recombinant human granulocyte-macrophage colony stimulating factor (hGM-CSF) from plant cell suspension culture in PEG/sodium phosphate aqueous two-phase systems

Partitioning of human granulocyte-macrophage colony stimulating factor (hGM-CSF) was achieved in the aqueous two-phase systems (ATPSs) using a crude extract of transgenic tobacco cell suspension culture. This study examined the effects of polyethylene glycol (PEG) molecular weight and concentration and the effects of sodium phosphate concentration in different PEG/sodium phosphate systems on the partition coefficient,K. The best ATPS system was 5% PEG 8,000/1.6 M sodium phosphate after 2 h of incubation at room temperature. In this system, hGM-CSF was partitioned in the PEG-rich phase with a yield of 57.99% andKhGM-CSF of 8.12. In another system, 3% PEG 10,000/1.6 M sodium phosphate, hGM-CSF was also partitioned primarily in the top phase with a yield of 45.66% andKhGM-CSF of 7.64 after 2 h of incubation at room temperature.

Texturization of monocrystalline silicon with tribasic sodium phosphate

Abstract Tribasic sodium phosphate (Na 3 PO 4 ·12H 2 O) was successfully introduced to texture monocrystalline silicon for the first time. A series of comparative experiments were made to indicate the dependence of hemispherical surface reflectance on the tribasic sodium phosphate (Na 3 PO 4 ·12H 2 O) concentration, reaction temperature and etching time. Meanwhile, the effects of other agents, such as isopropyl alcohol (IPA), sodium hydroxide (NaOH) and bi-sodium hydrogen phosphate (Na 2 HPO 4 ) on average reflectance were also investigated. The results showed that IPA and NaOH have great detrimental effects on texture, and the average reflectance slightly increased with the addition of Na 2 HPO 4 . On the basis of our experiments, it is concluded that the effect of phosphatidate (PO 4 3− ) or its compounds on the texturization is the same as that of the mixture of alkaline and IPA. Furthermore, this method is economical, has low pollution and good reproducibility. We feel that it is suitable for the large-scale production.

Partitioning of Ganglioside-Reconstituted Liposomes in Aqueous Two-Phase Systems

The partitioning of ganglioside (GM3,GD1a, GD1b or GT1b)-reconstituted liposomes was investigated in an aqueous poly(ethylene oxide)/dextran two-phase system to evaluate the carbohydrate–carbohydrate interactions in water. The partitioning of the ganglioside-reconstituted liposome was strongly affected by the buffer employed. As the concentration of sodium phosphate decreased, the ganglioside-reconstituted liposomes were partitioned to the bottom, dextran-rich, phase. In 10 mM sodium phosphate containing 150 mM sodium chloride, the conventional liposome without ganglioside were located mostly at the interface between the two phases. On the other hand, the ganglioside-reconstituted liposomes were significantly partitioned to the bottom dextran-rich phase, which was related to the ganglioside density on the liposomal surface. This partitioningto the dextran-rich phase also depended on the chemical structure of the ganglioside on the liposomal surface. The affinity of liposomal ganglioside being on the liposomal surface to dextran was the following sequence; GT1b>GD1a>GD1b>GM3. Partitioning of liposomes to the top poly(ethylene oxide)-rich phase was negligible irrespective of the characteristics of the liposomal surface.

Proteolytic Sensitivity and Helper T-cell Epitope Immunodominance Associated with the Mobile Loop in Hsp10s

Antigen three-dimensional structure potentially limits antigen processing and presentation to helper T-cell epitopes. The association of helper T-cell epitopes with the mobile loop in Hsp10s from mycobacteria and bacteriophage T4 suggests that the mobile loop facilitates proteolytic processing and presentation of adjacent sequences. Sites of initial proteolytic cleavage were mapped in divergent Hsp10s after treatment with a variety of proteases including cathepsin S. Each protease preferentially cleaved the Hsp10s in the mobile loop. Flexibility in the 22-residue mobile loop most probably allows it to conform to protease active sites. Three variants of the bacteriophage T4 Hsp10 were constructed with deletions in the mobile loop to test the hypothesis that shorter loops would be less sensitive to proteolysis. The two largest deletions effectively inhibited proteolysis by several proteases. Circular dichroism spectra and chemical cross-linking of the deletion variants indicate that the secondary and quaternary structures of the variants are native-like, and all three variants were more thermostable than the wild-type Hsp10. Local structural flexibility appears to be a general requirement for proteolytic sensitivity, and thus, it could be an important factor in antigen processing and helper T-cell epitope immunogenicity.

The effects of organic and inorganic phosphates on fertilization and early development in the sea urchin Lytechinusvariegatus (Echinodermata: Echinoidea)

The embryos of the sea urchin Lytechinus variegatus are capable of surviving chronic exposure to inorganic sodium phosphate and organic triethyl phosphate concentrations as high as 6 and 1000 mg l −1 seawater, respectively. However, chronic exposure to sub-lethal concentrations of these phosphates may cause arrested or abnormal embryonic development. We measured fertilization success and percentages of normal, arrested and abnormal embryos exposed to low, medium and high sub-lethal concentrations of inorganic and organic phosphate. Fertilization success was significantly reduced in all phosphate treatments. After attaining the 4-cell stage, embryos exposed to the highest phosphate concentrations displayed arrested development. Percentages of abnormally developing embryos showed a strong concentration dose–response with a significant increase in abnormal embryonic development with increasing phosphate concentration. Overall, these results indicate that the gametes and embryos of L. variegatus may provide a rapid and sensitive model bioassay for the evaluation of phosphate pollutants in marine systems. Our findings also indicate that shallow-water populations of L. variegatus spawning in areas subjected to inorganic and organic phosphate pollutants may suffer detrimental effects on fertilization and embryonic development.

Use of chromatography to investigate biological recognition of a ligand by human serum albumin

Chromatographic temperature studies and differential scanning calorimetry have been used to elucidate the mechanism of retention of dansyl amino acids by human serum albumin (HSA) as stationary phase in reversed-phase liquid chromatography (RPLC) (Guillaume et al.,Anal. Chem. 1997,69, 4979). A novel mathematical development is proposed for description of HSA-dansyl amino acid association when HSA is used as mobile-phase modifier and C18 as stationary phase. The solute retention factor depends on the concentration of HSA in the bulk mobile phase and on the binding constant for the guest-HSA complex. The degree of complexationn c (the amount, %, of guest complexed) was determined. Enthalpy-entropy compensation was also analyzed in relation to this mathematical model to confirm the complexation behavior of the solute with HSA. Variation of the binding constant with the concentration of sodium phosphate, i.e. with the electrostatic force controlling HSA-solute association, is described. This theoretical model enabled estimation of the surface charge density (σ/F) and the accessible solvent surface area of the site II binding cavity; these were found to be approximately 8.7×10−7 mol m−2 and 2 nm2.

Effects of inorganic and organic phosphates on feeding, feeding absorption, nutrient allocation, growth and righting responses of the sea urchin Lytechinus variegatus

The sea urchin Lytechinus variegatus is capable of surviving chronic exposure to sodium phosphate (inorganic phosphate) concentrations as high as 3.2 mg l−1, and triethyl phosphate (organic phosphate) concentrations of 1,000 mg l−1. However, chronic exposure to low (0.8 mg l−1 inorganic and 10 mg l−1 organic phosphate), medium (1.6 mg l−1 inorganic and 100 mg l−1 organic phosphate) or high (3.2 mg l−1 inorganic and 1,000 mg l−1 organic phosphate) sublethal concentrations of these phosphates inhibits feeding, fecal production, nutrient absorption and allocation, growth and righting behavior. Food consumption and fecal production declined significantly in individuals exposed to medium and high concentrations of inorganic phosphates and all levels of organic phosphates. Feeding absorption efficiencies for total organics and carbohydrates decreased significantly in individuals held in the highest concentration of organic phosphate. Feeding absorption efficiencies for lipids were significantly reduced in the highest inorganic phosphate concentration only, while they decreased significantly for protein with increasing phosphate exposure. Carbohydrate and lipid levels in gonad and gut tissues decreased significantly with exposure to increasing phosphate concentrations, potentially impairing both gametogenesis and nutrient storage in the gut. Moreover, gonad indices significantly decreased in individuals exposed to the highest concentrations of either phosphate. Growth rates decreased significantly under the influence of all phosphate concentrations, while increasing in seawater alone. Individuals exposed to increasing phosphate concentrations showed reduced righting responses (a measure of stress) and no acclimation in righting times during chronic exposure to phosphates over a 4 week period. These findings indicate that shallow-water populations of L. variegatus subjected to inorganic and organic phosphate pollutants will exhibit stress and be inhibited in their growth and performance due to reductions in feeding, nutrient absorption and allocation of nutrients to key somatic and reproductive tissues.

Trends in lipoplex physical properties dependent on cationic lipid structure, vehicle and complexation procedure do not correlate with biological activity.

Using a group of structurally related cytofectins, the effects of different vehicle constituents and mixing techniques on the physical properties and biological activity of lipoplexes were systematically examined. Physical properties were examined using a combination of dye accessibility assays, centrifugation, gel electrophoresis and dynamic light scattering. Biological activity was examined using in vitro transfection. Lipoplexes were formulated using two injection vehicles commonly used for in vivo delivery (PBS pH 7.2 and 0.9% saline), and a sodium phosphate vehicle previously shown to enhance the biological activity of naked pDNA and lipoplex formulations. Phosphate was found to be unique in its effect on lipoplexes. Specifically, the accessible pDNA in lipoplexes formulated with cytofectins containing a gamma-amine substitution in the headgroup was dependent on alkyl side chain length and sodium phosphate concentration, but the same effects were not observed when using cytofectins containing a beta-OH headgroup substitution. The physicochemical features of the phosphate anion, which give rise to this effect in gamma-amine cytofectins, were deduced using a series of phosphate analogs. The effects of the formulation vehicle on transfection were found to be cell type-dependent; however, of the formulation variables examined, the liposome/pDNA mixing method had the greatest effect on transgene expression in vitro. Thus, though predictive physical structure relationships involving the vehicle and cytofectin components of the lipoplex were uncovered, they did not extrapolate to trends in biological activity.

Factors Affecting Glucose and Maltose Phosphorylation by the Ruminal Bacterium Megasphaera elsdenii

The objectives of this study were to examine the effects of growth substrate and extracellular pH on phosphoenolpyruvate-dependent glucose phosphorylation as well as to examine how maltose is phosphorylated by the ruminal bacterium Megasphaera elsdenii B159. Phosphoenolpyruvate-dependent glucose phosphorylation by toluene-treated cells was constitutive, and glucose phosphorylation was reduced by 69% at pH 5.0. When toluene-treated cells were incubated in histidine buffer, little maltose phosphorylation occurred in the absence of inorganic phosphate. However, the addition of increasing concentrations of either potassium or sodium phosphate increased maltose phosphorylation. Maximal phosphorylation activity was observed at between 25 and 50 mM of either inorganic phosphate source. Compared with the control incubations, maltose phosphorylation was increased over threefold with 25 mM of either potassium or sodium phosphate. Phosphoglucomutase activity was detected in cell extracts of M. elsdenii B159, and this enzyme had a K(m) of 3.2 mM for glucose-1-P and a V(max) of 1836 nmol of NADP(+) reduced/mg of protein per min. Maltose was also hydrolyzed by an inducible maltase (K(m), 1.19 mM). To our knowledge, this is the first report of a maltose phosphorylase and a maltase in M. elsdenii.

Effects of Corticosteroid on Porcine Retinal Pigment Epithelial Cells in Culture: 1. Inhibitory Effect on Cell Proliferation

Purpose: Proliferation is an important function of the retinal pigment epithelial (RPE) cells. The effect of a corticosteroid on the proliferation of cultured porcine RPE cells was investigated. Methods: After administration of various concentrations (10–1,000 nM) of betamethasone sodium phosphate (betamethasone), we counted RPE cell numbers at 1, 3, 6, 9, and 14 days. Results: Betamethasone administration resulted in a dose-dependent decrease in RPE cell proliferation. The proliferation of the cultured RPE cells was significantly inhibited by betamethasone, at a dose of 300 nM in 9 days. Ten nanomolar betamethasone neither inhibited nor promoted the RPE cell proliferation in culture. Conclusions: These results suggest that corticosteroids inhibit proliferation of cultured porcine RPE cells. (J Jpn Ophthalmol Soc 103:436–441, 1999)

Apolipoprotein A-I(Milano) unfolds via an intermediate state as studied by differential scanning calorimetry and circular dichroism.

In this study the thermal and denaturant induced denaturation behaviors of apolipoprotein A-I(Milano) (apo A-IM) have been studied by differential scanning calorimetry and circular dichroism spectroscopy, as well as solution properties by analytical ultracentrifugation. Thermal denaturation is dependent on pH, sodium phosphate concentration and NaCl concentration. The protein is highly self-associated at the protein concentrations used in this study. Denaturation of apo A-IM at pH 7.4 and 8.0 occurs in two steps. The midpoint between the transition is at 37 degrees C. The first step at 31 degrees C involves melting of tertiary structure and rearrangement of protein association complexes, i.e. a transition into an intermediate molten globular-like state. Subsequent melting of this intermediate state into an unfolded state occurs at 52 degrees C. At pH 2.8 the protein lacks all tertiary structure and denaturation occurs over a large temperature interval, indicating the induction of a molten globular-like state at low pH.

Simultaneous Determination of Trace Oxoanions Using Ion Chromatography With Ultraviolet Absorbance Detection

CS5A column had bifunctional (cation exchange/anion exchange) stationary phase. It was the first time that CS5A column was used for the analysis of trace oxoanions by ion chromatography. The influence of sodium phosphate concentration, methanol concentration, and eluent pH on their separations were studied. An eluent of 7.5 mM Na2HPO4, pH 9.3 was found to be the most suitable for the analysis of SeO3 2−, HAsO4 2−, SeO4 2−, WO4 2−, MoO4 2−, GeO3 2−, and CrO4 2− simulanteously. An eluent of 0.75 mM Na2HPO4, pH 9.3 was found to be the most suitable for the analysis of IO3 −, H2AsO3 −, BrO3 −, NO2 −, and NO3 − simulanteously. The detection method employed was selective and direct UV absorbance at 204 nm was utilized. Several other anion separation columns (AS4A-SC column, AS9-SC column, AS11 column, and PAX 500 column), which had different structures and properties, were compared to CS5A column. Among them, CS5A column was the best choice.

Human cytochrome P450 3A (CYP3A) mediated midazolam metabolism

The effect of ionic strength, assay constituents, alpha-naphthoflavone (aNF), terfenadine and testosterone on human CYP3A mediated midazolam (MDZ) 1'-hydroxylation (MDZ 1'-OH) and 4-hydroxylation (MDZ 4-OH) in vitro was examined. Increasing concentration of Tris-HCl (Tris) and sodium phosphate (PO4) buffers differentially affected MDZ 1'-OH and MDZ 4-OH formation rates and had a different effect on MDZ metabolism mediated by microsomes containing CYP3A4 versus CYP3A4 and CYP3A5. MDZ metabolism was not affected by PO4 buffer concentration when cumene hydroperoxide (CUOOH) was used as the source of reactive oxygen. Interestingly, the ammonium ion present in the solution of glucose 6-phosphate dehydrogenase was found to inhibit MDZ metabolism. The addition of MgCl2 up to 50 mM and CaCl2 (5-30 mM) had no affect or inhibited MDZ metabolism, respectively. Formation of MDZ 1'-OH by microsomes from adult and fetal liver and expressed CYP3A4 was regioselectively stimulated by aNF (10 microM). In human hepatocytes, aNF stimulated MDZ 1'-OH formation (up to 100%). Terfenadine (20 microM) regioselectively stimulated MDZ 1'-OH formation in Tris (1-200 mM) and PO4 (1-10 mM) buffers by up to 159%. Surprisingly, with expressed CYP3A4, terfenadine (20 microM) inhibited MDZ 1'-OH formation. Terfenadine (20 microM) had little effect on MDZ 1'-OH formation by fetal liver microsomes. Testosterone (10 and 100 microM) regioselectively stimulated (up to 269%) MDZ 4-OH formation by adult liver microsomes and expressed CYP3A4. Testosterone (100 microM) inhibited (> 40%) MDZ 1'-OH and MDZ 4-OH formation by fetal liver microsomes. With adult liver microsomes, aNF and terfenadine had little effect on the Km for MDZ 1'-OH formation. However, the Km for MDZ 4-OH formation was decreased (up to 94%) by 100 microM testosterone. In the presence of CUOOH, no stimulation of MDZ metabolism was observed by aNF, terfenadine or testosterone in adult liver microsomes. These studies indicate that because assay conditions can substantially alter the catalytic activity of CYP3A, caution should be exerted when extrapolating results between in vitro and in vivo, and when results from different laboratories are compared. Further, these results suggest that the stimulation of CYP3A4 may also occur in vivo and, consequently, may have clinical importance.

Glyphosate Uptake inCatharanthus roseusCells: Role of a Phosphate Transporter

InCatharanthus roseusL. cells, the study of glyphosate uptake indicates that, at low concentrations, glyphosate is transported against a concentration gradient, reaching an accumulation ratio of about 30, 18 h after the cells are transferred into a fresh medium. Evidence that part of the glyphosate uptake may be carrier mediated was provided by concentration dependence experiments which showed that glyphosate uptake exhibited a saturation phase at low concentrations (up to 50 μM). The role played by a phosphate transporter in this process is demonstrated by the existence of a lag period and the inhibition of glyphosate uptake in the presence of high concentrations of sodium phosphate. It is also shown by the effect of PFA, a powerful inhibitor of phosphate transport in animal cells, and also by the action of protein chemical reagents (PCMBS and DCCD). Compartmental analysis with isolated protoplasts and vacuoles indicated that [14C]glyphosate is distributed between the cytosolic and the vacuolar compartments, but the greater part is localized in the cytosol. Detailed studies carried out to investigate the requirements of the glyphosate transporter showed that, among the different constituents of the Gamborg's nutrient medium, the major elements increasing the cellular glyphosate uptake were Ca2+, Mg2+, and the presence of iron.