Fe3+ binding to ovotransferrin in the presence of alpha-amino acids.

The ability of L-alpha-amino acids as synergistic anions for iron binding to ovotransferrin was investigated through electronic spectroscopy. Glycine and glutamic acid were found to form by far the most stable ternary Fe(3+)-ovotransferrin-amino acid complexes. Less stable adducts were formed by amino acids with a hydroxy, amide or sulphur-containing group in the side chain, while the complexes with leucine, isoleucine, valine, lysine, arginine, tyrosine and tryptophan failed to form. Evidence is obtained that the synergistic effectiveness of the H2N-CH-COO- moiety is determined not only by the isoelectric point of the amino acid and the steric hindrance of its side chain, but a significant role is also played by interactions of the side chain itself with residues in the metal binding domains. Zn2+, Cd2+ and Co2+ are found to bind to ovotransferrin in the presence of glycine. 113Cd-NMR spectra on the Cd-derivative indicate that, according to the interlocking-sites model, the amino group of glycine directly binds to the metal ion.     

Al3+ versus Ca2+ ion binding to methionine and tyrosine spin-labeled bovine brain calmodulin.

Bovine calmodulin analogues, spin-labeled at either methionine or tyrosine residues, have been utilized in electron paramagnetic resonance (EPR) studies to investigate possible calmodulin interactions with aluminum ion. The study attempts to clarify a previous report in the literature (H. Siegel, R. Coughlin, and A. Haug, Biochem. Biophys. Res. Commun. 115, 512 (1983)) which indicated, on the basis of EPR experiments on methionine spin-labeled protein, significant interaction between calmodulin and aluminum ion at pH = 6.5. In EPR metal ion titration experiments we have found that the signal line-shape (from both methionine and tyrosine spin labels) changed dramatically with the addition of calcium ion, but was virtually unchanged with the addition of aluminum ion at pH = 6.5. Experiments performed at pH = 5.5, where significantly more "free" aluminum ion (i.e., Al(H2O)6(3+) = Al3+) is present, also failed to produce the line-narrowing effect observed in the earlier study. Based on our EPR experiments, in the pH range 5.5 to 6.5, we find no evidence for significant interaction between calmodulin and aluminum ion.     

Regulation of Fe3+-oxide Formation Among Fe2+-oxidizing Bacteria

Helical stalks (resembling Gallionella ferruginea, Mariprofundus ferrooxydans) and filamentous sheaths (resembling Leptothrix ochracea) of Fe²⁺-oxidizing bacteria (FeOB) are mineralized by hydrous ferric oxides (HFO). To perform both inter-species and inter-site size comparisons of HFO particles on stalks and sheaths we measured HFO particles in samples of natural bacteriogenic iron oxides (BIOS) from 3 contrasting field sites: the Loihi Seamount (southern Hawaii); Äspö Hard Rock Laboratory (eastern Sweden); and Chalk River Laboratories (northern Canada) representing seafloor saline, underground brackish, and surface freshwater aqueous conditions. Ambient temperatures were in the psychrophilic range and pHs measured for Loihi, CRL, and Äspö were 5.6, 6.9 and 7.4, respectively. Dissolved Fe was lowest for CRL (0.2 mg · L^?1) followed by Äspö (1.5 mg · L^?1), then Loihi (4.5–14.9 mg · L^?1). L. ochraceasheaths appear to have surface properties that restrict HFO particle growth in comparison to G.ferruginea-M.ferrooxydans stalks in the same environment, which we attribute to interfacial surface energy (γ). An inverse relationship between particle size and stalk/sheath length due to restrictions in reactive surface area was also observed, which may provide insight into FeOB survival strategies to alleviate oxidative stress arising from Fe³⁺ production.     

Fe2+ binding to apo and holo mammalian ferritin

The binding of Fe2+ to both apo and holo mammalian ferritin has been investigated under anaerobic conditions as a function of pH. In the pH range 6.0-7.5, 8.0 +/- 0.5 Fe2+ ions bind to each apoferritin molecule, but above pH 7.5, a pH-dependent Fe2+ binding profile is observed with up to 80 Fe2+ ions binding at pH 10.0. This Fe2+ binding is reversible and is accompanied by up to two H+ being released per Fe2+ bound at pH 10.0. The Fe2+ binding to apoferritin probably occurs in the 3-fold channels. A much larger and more complex pH-dependent Fe2+ binding stoichiometry was observed for holoferritin with up to 300 Fe2+ ions binding at pH 10.0. This pH-dependent Fe2+ binding was interpreted as Fe2+ interaction at the FeOOH mineral surface with displacement of H+ from -OH or phosphate surface groups by the incoming Fe2+ ions. Mossbauer spectroscopic measurements using 57Fe-labeled Fe2+ under anaerobic conditions showed that 57Fe2+ binding to holoferritin was accompanied by electron transfer to the core, yielding 57Fe3+, presumably bound to the mineral surface. Removal of added iron by Fe2+-specific chelating agents yielded 57Fe2+, demonstrating the reversibility of this electron-transfer process. The Fe2+ bound to apo- and holoferritin is readily converted to Fe3+ by exposure to O2 and strongly retained by the respective ferritin species.     

The nonspecific binding of Fe3+ to transferrin in the absence of synergistic anions.

An obligatory role for barbonate (or other synergistic anions) in the specific binding of Fe3+ by transferrin has been a point of controversy for two decades. There are an equal number of confirmatory and negative reports of specific Fe3+-transferrin binary complexes. A criticism of previous studies is the use of only one synthetic route, and limited product testing. This study reports the development of several preparative routes aimed at the formation of a specific Fe3+-transferrin complex, and the characterization of the products by spectrophotometry and chemical reactivity. The preparative routes described include: (a) displacement of carbonate from Fe3+-transferrin-CO32- at low pH followed by removal of CO2 by several techniques; (b) addition of FeCl3 to apotransferrin under CO2-free conditions; (c) oxidation of Fe2+ in the presence of apotransferrin under CO2-free conditions; (d) reaction of apotransferrin with nonsubstituting Fe3+ complexes in the absence of CO2; and (e) attempts to displace anions from weak Fe3+-transferrin-anion complexes. The product were examined with regard to their visible spectra, and their examined with regard to their visible spectra, and their reactivity with: (a) NaHCO3, (b) Fe3+-nitrilotriacetic acid in NaHCO3, and (c) citrate. The results are compared with the characteristics of Fe3+-transferrin-anion complexes and nonspecific Fe3+, transferrin mixtures. The data indicate that in the absence of synergistic anions the affinity of the specific metal binding sites of transfe-rin for Fe3+ is so low as to not compete favorably with hydrolytic polymerization and nonspecific binding effects.     

Serotonin binding protein:enhancement of binding by Fe2+ and inhibition of binding by drugs.

The binding of serotonin to a soluble, high affinity binding protein, present in synaptosomes and associated with serotonergic tracts, has now been studied for the effects of metallic ions and various drugs. At optimal concentration (10^-4 M) of Fe²⁺ the enhancement of binding was close to 20-fold. A much smaller effect was noted with Cu²⁺. With other ions (Fe³⁺, Mn²⁺, Co²⁺, Ni²⁺, Cr³⁺, Mg²⁺, Ca²⁺) little or no effect was seen. For the effect with Fe²⁺. preincubation was required (10 min, 25°C) and concentrations higher than 10^-4M were inhibitory. Studies based on equilibrium dialysis show that the effect of Fe²⁺ was on the affinity of the binding of serotonin to the protein, rather than on the binding capacity. In polydcrylamide gels at pH 8.6 the migratory properties of thc serotonin-protein complex formed in the presence of Fe²⁺ differ from those of the complex formed without Fe²⁺. Nucleotides (ATP, GTP, ADP, AMP) inhibited thc binding. The effects of several classes of drugs (inhibitors of biogenic amine storage and uptake, psychotomimetics, MAO) inhibitors and drugs binding to contractile proteins) were also studied. The only effective inhibitors of serotonin binding were reserpine, vinblastine and CZ-74, which caused 50% inhibition at 2 × 10^-6 M, 7.5 × 10^-6 M and 0.2 × 10^-6M respectively.     

Al3+-Ca2+ interactions in aluminum rhizotoxicity

Several mineral rhizotoxicities, including those induced by Al³⁺, H⁺, and Na⁺, can be relieved by elevated Ca²⁺ in the rooting medium. This leads to the hypothesis that the toxic cations displace Ca²⁺ from transport channels or surface ligands that must be occupied by Ca²⁺ in order for root elongation to occur. In this study with wheat (Triticum aestivum L.) seedlings, we have determined, in the case of Al³⁺, that (i) Ca²⁺, Mg²⁺, and Sr²⁺ are equally ameliorative, (ii) that root elongation does not increase as Ca²⁺ replaces Mg²⁺ or Sr²⁺ in the rooting media, and (iii) that rhizotoxicity is a function solely of Al³⁺ activity at the root-cell membrane surface as computed by a Gouy-Chapman-Stern model. The rhizotoxicity was indifferent to the computed membrane-surface Ca²⁺ activity. The rhizotoxicity induced by high levels of tris(ethylenediamine)cobaltic ion (TEC³⁺), in contrast to Al³⁺, was specifically relieved by Ca²⁺ at the membrane surface. The rhizotoxicity induced by H⁺ exhibited a weak specific response to Ca²⁺ at the membrane surface. We conclude that the Ca²⁺-displacement hypothesis fails in the case of Al³⁺ rhizotoxicity and that amelioration by cations (including monovalent cations) occurs because of decreased membrane-surface negativity and the consequent decrease in the membrane-surface activity of Al³⁺. However, TEC³⁺, but not Al³⁺, may be toxic because it inhibits Ca²⁺ uptake. The nature of the specific H⁺-Ca²⁺ interaction is uncertain.Abbreviations {Al³⁺ }₀ chemical activity of Al³⁺ at the root-cell membrane surface - {Al³⁺ }_E chemical activity of Al³⁺ in the external rooting medium - E₀ electrical potential at the root-cell membrane surface - HXM²⁺ hexamethonium ion - TEC³⁺ tris(ethylenediamine)cobaltic ion     

Simultaneous Overexpression of Citrate Synthase and Phosphoenolpyruvate Carboxylase in Leaves Augments Citrate Exclusion and Al Resistance in Transgenic Tobacco

Phosphoenolpyruvate carboxylase (PEPC) and citrate synthase (CS) are two key enzymes in organic acid synthesis metabolism. In the present study, a cytoplasmic form of CS from tobacco and a mutant (with reduced sensitivity to organic acid inhibition) PEPC from Synechococcus vulcanus were overexpressed simultaneously using a light-inducible promoter in tobacco leaves. The analysis for enzyme activity showed that CS and PEPC enzyme activities were increased by 235% to 257% and 218% to 236% in the selected cs and pepc (double-gene) overexpression lines, respectively, compared with those in the wild-type plants (WT). The measurement for the relative root elongation rate of the tobacco plants exposed to 30???M aluminum (Al) indicated that Al tolerance in the double-gene overexpression lines was stronger than that of the transgenic cs or pepc lines and WT plants. The ¹³C-NMR analysis with NaH¹³CO₃ showed that overexpression of CS and PEPC in the transgenic tobacco successfully constructed a new citrate synthesis pathway. Under the conditions with Al stress, the amount of citrate secreted from the double-transgenic tobacco roots was the largest among the tested plants. When grown on sandy soil supplied with a nutritional solution containing 500???M Al, the growth of the double-transgenic tobacco was better than that of the transgenic cs or pepc tobacco and WT, and their root biomass was the highest among the tested plants. These results demonstrated that construction of a new citrate synthesis pathway by simultaneous overexpression of CS and PEPC in the cytoplasm of transgenic plant leaves could enhance Al resistance in plants.     

Al(3+) interaction sites of calmodulin and the Al(3+) effect on target binding of calmodulin

The interaction between calmodulin (CaM) and Al(3+) was studied by spectroscopic methods. Heteronuclear two-dimensional NMR data indicated that peaks related to the both lobes and middle of the central helix of CaM are largely affected by Al(3+). But chemical shift perturbation suggested that overall conformation of Ca(2+)-loaded CaM is not changed by Al(3+) binding. It is thought that Al(3+) interaction to the middle of the central helix is a key for the property of CaM's target recognition. If the structure and/or flexibility of the central helix are/is changed by Al(3+), target affinity to CaM must be influenced by Al(3+). Thus, we performed surface plasmon resonance experiments to observe the effect of Al(3+) on the target recognition by CaM. The data clearly indicated that target affinity to CaM is reduced by addition of Al(3+). All the results presented here support a hypothesis that Al(3+) may affect on the Ca(2+) signaling pathway in cells.     

Fe3+ coordination and redox properties of a bacterial transferrin

The Fe(3+) binding site of recombinant nFbp, a ferric-binding protein found in the periplasmic space of pathogenic Neisseria, has been characterized by physicochemical techniques. An effective Fe(3+) binding constant in the presence of 350 microm phosphate at pH 6.5 and 25 degrees C was determined as 2.4 x 10(18) m(-1). EPR spectra for the recombinant Fe(3+)nFbp gave g' = 4.3 and 9 signals characteristic of high spin Fe(3+) in a strong ligand field of low (orthorhombic) symmetry. (31)P NMR experiments demonstrated the presence of bound phosphate in the holo form of nFbp and showed that phosphate can be dialyzed away in the absence of Fe(3+) in apo-nFbp. Finally, an uncorrected Fe(3+/2+) redox potential for Fe-nFbp was determined to be -290 mV (NHE) at pH 6.5, 20 degrees C. Whereas our findings show that nFbp and mammalian transferrin have similar Fe(3+) binding constants and EPR spectra, they differ greatly in their redox potentials. This has implications for the mechanism of Fe transport across the periplasmic space of Gram-negative bacteria.     

Molecular and Physiological Analysis of Al3+ and H+ Rhizotoxicities at Moderately Acidic Conditions

Al³⁺ and H⁺ toxicities predicted to occur at moderately acidic conditions (pH [water] = 5–5.5) in low-Ca soils were characterized by the combined approaches of computational modeling of electrostatic interactions of ions at the root plasma membrane (PM) surface and molecular/physiological analyses in Arabidopsis (Arabidopsis thaliana). Root growth inhibition in known hypersensitive mutants was correlated with computed {Al³⁺} at the PM surface ({Al3+}_PM); inhibition was alleviated by increased Ca, which also reduced {Al3+}_PM and correlated with cellular Al responses based on expression analysis of genes that are markers for Al stress. The Al-inducible Al tolerance genes ALUMINUM-ACTIVATED MALATE TRANSPORTER1 and ALUMINUM SENSITIVE3 were induced by levels of {Al3+}_PM too low to inhibit root growth in tolerant genotypes, indicating that protective responses are triggered when {Al3+}_PM was below levels that can initiate injury. Modeling of the H⁺ sensitivity of the SENSITIVE TO PROTON RHIZOTOXICITY1 knockout mutant identified a Ca alleviation mechanism of H⁺ rhizotoxicity, possibly involving stabilization of the cell wall. The phosphatidate phosphohydrolase1 (pah1) pah2 double mutant showed enhanced Al susceptibility under low-P conditions, where greater levels of negatively charged phospholipids in the PM occur, which increases {Al3+}_PM through increased PM surface negativity compared with wild-type plants. Finally, we found that the nonalkalinizing Ca fertilizer gypsum improved the tolerance of the sensitive genotypes in moderately acidic soils. These findings fit our modeling predictions that root toxicity to Al³⁺ and H⁺ in moderately acidic soils involves interactions between both toxic ions in relation to Ca alleviation.Aluminum (Al), principally in the form of Al³⁺ released from soil clay minerals, is one of the most important rhizotoxic ions in acidic soils and is abundant in soil solutions at pH (water) of less than 5.0. Many forest and grass land species naturally adapted to acid soils are very tolerant of H⁺ and Al³⁺ and thrive in soils where the pH is less than 4.0. However, most crop plants used for agriculture show inhibitory growth effects, even when the soil pH is neutralized by liming to moderately acidic pH values in the range of pH 5 to 5.5. For example, crops sensitive to Al³⁺ and H⁺ such as turnip (Brassica rapa; Kinraide and Parker, 1990) and alfalfa (Medicago sativa; Yokota and Ojima, 1995) show growth inhibition at these soil pH values. Field research and soil experiments have shown that inhibitory effects of moderately acidic soils (pH > 5) can be ameliorated by the application of Ca fertilizers, even if they are nonalkalinizing, such as gypsum, and this leads to improvement in crop productivity (Carvalho and Van Raij, 1997; Mora et al., 1999). This indicates that the soil Ca status is an important factor in determining crop yield at moderately low soil pH values with regard to Al³⁺ and H⁺ rhizotoxicity occurring in these soils. An understanding of the complex situation of acid soil stress in soil pH in the range of pH (water) 5 to 5.5 is important for designing efficient soil acidity management and breeding programs for resistant crop use in low-input agricultural systems.The complex rhizotoxicities at moderately acidic conditions that can be alleviated by Ca have been predicted by modeling studies in wheat (Triticum aestivum; Kinraide, 2003). The model first computes the activity of the rhizotoxicants and alleviants at the plasma membrane (PM) surface, for example {Al3+}_PM and {H+}_PM, and {Ca2+}_PM, using a speciation-based Gouy-Chapman-Stern electrostatic (SGCS) model (Kinraide and Wang, 2010). The mechanisms of toxicity and alleviation are then modeled by regression analyses for root growth inhibition fitted to the nonlinear equations (Kinraide, 2003; Kinraide et al., 2004). The modeling studies well describe the complicated events in the Al-toxic solutions near the PM surface under moderately acidic conditions. For example, the elevation of pH from 4.5 to between 5 and 5.5 decreases the activity of the most rhizotoxic Al species, Al³⁺ in the solution ({Al3+}_bulk), while it increases the negativity of the PM surface because of dissociation of H⁺ from potentially negative ligands such as phospholipids. As a result, {Al3+}_PM remains at moderately high levels at the PM surface at pH greater than 5 due to the attraction to negative charges on the PM surface, but it can be alleviated by coexisting cations such as Ca²⁺ and even by another rhizotoxic cation, H⁺. These modeling studies have proposed different mechanisms of Ca alleviation in this complex situation (Kinraide, 1998; Kinraide et al., 2004). Mechanisms I (the electrostatic displacement of toxicant at the PM surface) and II (the restoration at the PM surface of Ca²⁺ electrostatically displaced by the toxicant) are events at the PM surface, but mechanism III, which explains the remaining portion of the Ca alleviation, may involve other physiological responses, including unknown mechanisms. These predictions, derived from the modeling study, likely explain the complex nature of moderately acidic soils but may require further validation because they were developed using root growth as the sole criterion for rhizotoxicity.Although these types of modeling approaches have not been performed using Arabidopsis (Arabidopsis thaliana) plants, clear symptoms of Al³⁺ and H⁺ rhizotoxicity at moderately acidic conditions (pH ≥ 5) has been identified in Arabidopsis (Kobayashi and Koyama, 2002; Iuchi et al., 2007). A quantitative trait locus study of Al tolerance at moderately acidic conditions (4 μm Al, pH 5; Kobayashi and Koyama, 2002) identified a very similar genetic architecture of Al tolerance to that derived from a study that employed a lower pH value but with a greater level of Al (50 μm Al, pH 4.2; Hoekenga et al., 2003). The former conditions employed a lower Ca concentration (200 μm) than the latter (3 mm), which accounted for the predictions of {Al3+}_PM in relation to {Ca2+}_PM by electrostatics. On the other hand, several Al³⁺- and H⁺-sensitive transfer DNA insertion knockout (KO) mutant genotypes have been identified using the lower ionic-strength moderately acidic media (Sawaki et al., 2009). These lines exhibit different degrees of hypersensitivity to moderately acidic conditions because of the dysfunction of different tolerance genes, suggesting the involvement of different mechanisms. In Arabidopsis, ALUMINUM-ACTIVATED MALATE TRANSPORTER1 (AtALMT1) regulates Al-activated root malate excretion that protects the root tip from acute Al toxicity by Al exclusion (Hoekenga et al., 2006), and ALUMINUM SENSITIVE3 (ALS3) regulates internal Al sequestration involved in long term Al tolerance (Larsen et al., 1997, 2005). The KO mutants for these genes display Al hypersensitivity. In addition, SENSITIVE TO PROTON RHIZOTOXICITY1 (STOP1)-KO, a suppressor of multiple genes for Al and H⁺ tolerance, shows sensitivity to Al³⁺ and H⁺ (Iuchi et al., 2007; Sawaki et al., 2009). These sensitive genotypes are useful tools for evaluating Al³⁺ and H⁺ toxicity in the pH range 5 to 5.5. On the other hand, several cellular responses, such as the induction of gene expression, have been identified in Arabidopsis that could be useful in the estimation of the attraction of {Al³⁺} to the PM, which is computed by our electrostatic-based model. Therefore, Arabidopsis appears to be a useful model system for the validation of modeling based on the SGCS model and to further our understanding of Al³⁺ and H⁺ rhizotoxicities at moderately acidic conditions in relation to Ca²⁺ alleviation.Computation of {Al3+}_PM requires accurate speciation of Al and other solutes in the bulk solution. The original SGCS program is suitable for relatively simple solutions (Kinraide and Wang, 2010). However, the rooting medium used for the Arabidopsis assays exceeds the number of solutes that can be accurately assessed by the SGCS program (Kobayashi et al., 2007). Consequently, we updated the modeling methodology using the speciation program GEOCHEM-EZ, which is suitable for complex media (Famoso et al., 2010; Shaff et al., 2010). This improved model, used in conjunction with molecular biological assays such as biomarker analysis of Al-inducible gene expression, has allowed us in this study to validate the predicted {Al3+}_PM rhizotoxicity in relation to {Ca2+}_PM alleviation from the wheat modeling studies. The updated modeling of Ca alleviation in mutants uncovered one of the mechanisms of Ca alleviation in the H⁺-sensitive mutant and identified an Al-sensitive double mutant genotype, phosphatidate phosphohydrolase1 (pah1) and pah2 (Nakamura et al., 2009), that fitted previous predictions. Finally, we demonstrate the ability of gypsum to ameliorate the sensitive phenotype of selected genotypes, when they were grown in moderately acidic soil culture. Taken together, we present here experimental validation of the SGCS-based modeling, and its combination with molecular physiology provides a deeper understanding of plant Al³⁺ and H⁺ toxicity in relation to Ca²⁺ alleviation at pΗ of at least 5.0.     

The X-ray structure of the pyochelin Fe3+ complex

By X-ray structure analysis it could be shown that from the solution equilibrium of pyochelin I and II, differing in the stereochemistry at C-2" (1a and 1b), crystals of the Fe3+ complex of the steroisomer 1a are formed with a 1:1 metal-to-ligand ratio. Ligand sites are the carboxylate and the phenolate anions and the two nitrogen atoms. Two equivalent ferri-pyochelin moieties are held together by a hydroxy and an acetate unit which satisfy the remaining two coordination sites of Fe3+.     

The NADH-dependent Fe3+-chelate reductases of tomato roots

The NADH-dependent Fe³⁺-chelate reductase (NFCHR) of tomato (Lycopersicon esculentum L.) roots, a strategy I species, was investigated. The Fe³⁺-citrate reductase (FeCitR) assay was strongly inhibited by p-hydroxymercuribenzoic acid (PHMB); moreover, the inhibitor was found to be more specific to the FeCitR assay than to the Fe³⁺-EDTA reductase assay, which was catalyzed by at least another reductase of 46 kDa. After high-speed centrifugation of tomato root membranes, high FeCitR activities were detected in pellets and lower activities in supernatants. After two-phase partitioning of microsomes, FeCitR activity (91 nmol · min⁻¹ · mg⁻¹) was less active in the upper phase (plasma membrane) than in the lower phase (277 nmol · min⁻¹ · mg⁻¹). However, only the activity of the plasma-membrane-associated NFCHR (FeCitR) was significantly enhanced (2.6-fold) in iron-deficient tomato plants, whereas that of NFCHR in non-plasma-membrane rich fractions was unaffected by this treatment. The NFCHR obtained from lysophosphatidylcholine-solubilized plasma membrane was present as a 200-kDa protein complex following fast protein liquid chromatography on Superdex 200, or as a 28-kDa form following Blue Sepharose CL-6B chromatography. Both preparations were more active following iron starvation. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis showed that the 28-kDa protein purified from solubilized tomato microsomes or supernatant fractions by a final Mono Q step consisted of a single band of 32 kDa. Tomato root NFCHR resembled the NFCHR of maize (a strategy II plant, P Bagnaresi and P Pupillo, 1995, J Exp Bot 46: 1497–1503) in several properties: relative molecular mass, hydrophilicity, chromatographic behaviour, sensitivity to mercurials, specificity for electron donors and acceptors (e.g. cytochrome c), and a ferricyanide reductase-to-FeCitR ratio of 2.5. Preincubation with NADH partially protected NFCHR from PHMB-induced inactivation. Our data show that strategy I and II plants seem to share similar NFCHR proteins, which appear to belong to the cytochrome b ₅ reductase flavoprotein group. Received: 6 November 1996 / Accepted: 21 January 1997     

Competition of Mn2+ and Zn2+ with59Fe2+ and59Fe3+ for the plasma membrane receptors from lactating mouse mammary gland

Nonlabeled MnCl₂ and ZnSO₄ compete with⁵⁹Fe²⁺-ascorbate and⁵⁹Fe ₂ ³⁺ O₃ for transport binding sites situated on the plasma membranes of lactating mouse mammary gland cells. The binding was found to be a process reaching saturation. The heterologous competition used here ruled out the participation of transferrin and to propose that Fe, Mn, and Zn are transported from blood to milk by a mechanism involving one receptor during lactation. Further experiments are necessary to establish the details of the transport mechanism.     

Fe and Ca Uptake as Related to Root-Sap and Stem-Exudate Citrate in Soybeans

Two soybean varieties that differentially absorb and translocate iron were used to compare root-sap citrate with stem-exudate citrate as they are involved in the uptake of Fe and Ca. The status of Fe and PO₄ in the prenutrient solution determined the citrate concentration in the root sap and the citrate translocated in the stem exudate. There was a parallel between the iron and the citrate translocated in the stem exudate, but this relationship did not appear to exist for the citrate and Fe concentrations in the root sap. Iron stress (deficiency) promoted the accumulation of citrate in the root-sap, but there was not a concomitant increase of citrate in the stem exudate. In iron-deficient soybeans, phosphate stress also promoted the accumulation of citrate in the root sap, and here, stem-exudate citrate and root-sap citrate more nearly followed the same trends. The citrate pool in the root appears to result from a deficiency of iron and may not be directly involved in the absorption and translocation of iron from the growth medium. Increasing amounts of phosphate in the prenutrient decreased both the citrate and Fe in the root sap and stem exudate. The factors controlling the uptake of Fe are rather specific and are not related to the uptake of radioactive Ca 45 in soybeans regardless of soybean variety, degree of iron stress, or citrate concentration in the root.     

Ternary hydroxide complexes in neutral solutions of Al3+ and F-

Especially in G protein systems AlF4- has been claimed as an activating species serving as a tetrahedral phosphate analog. However, in aqueous solutions (H2O)2AlF4- is hexacoordinate with two bound water molecules. In neutral solutions five different mixed OH- and F- complexes of Al3+ comprise the main species under usual experimental conditions. Comparison of the mole fraction distribution curves with limited results on the activity as a function of ambient F- concentrations suggests an activating complex composed of Al3+ with three F- and uncertain geometry. Even fewer activity data suggest a tetrahedral Be2+ complex with three F-.