The involvement of iron in lipid peroxidation. Importance of ferric to ferrous ratios in initiation

Intense lipid peroxidation of brain synaptosomes initiated with Fenton's reagent (H2O2 + Fe2+) began instantly upon addition of Fe2+ and preceded detectable OH. formation. Although mannitol or Tris partially blocked peroxidation, concentrations required were 10(3)-fold in excess of OH. actually formed, and inhibition by Tris was pH dependent. Lipid peroxidation also was initiated by either Fe2+ or Fe3+ alone, although significant lag phases (minutes) and slowed reaction rates were observed. Lag phases were dramatically reduced or nearly eliminated, and reaction rates were increased by a combination of Fe3+ and Fe2+. In this instance, lipid peroxidation initiated by optimal concentrations of H2O2 and Fe2+ could be mimicked or even surpassed by providing optimal ratios of Fe3+ to Fe2+. Peroxidation observed with Fe3+ alone was dependent upon trace amounts of contaminating Fe2+ in Fe3+ preparations. Optimal ratios of Fe3+:Fe2+ for the rapid initiation of lipid peroxidation were on order of 1:1 to 7:1. No OH. formation could be detected with this system. Although low concentrations of H2O2 or ascorbate increased lipid peroxidation by Fe2+ or Fe3+, respectively, high concentrations of H2O2 or ascorbate (in excess of iron) inhibited lipid peroxidation due to oxidative or reductive maintenance of iron exclusively in Fe2+ or Fe3+ form. Stimulation of lipid peroxidation by low concentrations of H2O2 or ascorbate was due to the oxidative or reductive creation of Fe3+:Fe2+ ratios. The data suggest that the absolute ratio of Fe3+ to Fe2+ was the primary determining factor for the initiation of lipid peroxidation reactions.     

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.     

Oxidation of ferrous iron during peroxidation of lipid substrates

Oxidation of Fe2+ in solution was dependent upon medium composition and the presence of lipid. The complete oxidation of Fe2+ in 0.9% saline was markedly accelerated in the presence of phosphate or EDTA and the ferrous oxidation product formed was readily recoverable as Fe2+ by ascorbate reduction. In contrast, in the presence of either brain synaptosomal membranes, phospholipid liposomes, fatty acid micelles or H2O2, less than 50% of the Fe2+ oxidized during an incubation could be recovered as Fe2+ via reduction with ascorbate. In the presence of unsaturated lipid, oxidation of Fe2+ was associated with peroxidation of lipid, as assessed by the uptake of O2 and formation of thiobarbituric acid-reactive products during incubations. Although relatively little Fe2+ oxidation or lipid peroxidation occurred in saline with synaptosomes or linoleic acid micelles during an incubation with Fe2+ alone, significant Fe2+ oxidation and lipid peroxidation occurred in incubations containing a 1:1 ratio of Fe2+ and Fe3+. Extensive Fe2+ oxidation and lipid peroxidation also occurred with Fe2+ alone in saline incubations with either linolenic or arachidonic acid acid micelles or liposomes prepared from dilinoleoylphosphatidylcholine. While a 1:1 ratio of Fe2+ and Fe3+ enhanced thiobarbituric acid-reactive product formation in incubations containing linolenic or arachidonic micelles, it reduced the rate of O2 consumption as compared with Fe2+ alone. The results demonstrate that oxidation of Fe2+ in incubations containing lipid substrates is linked to and accelerated by peroxidation of those substrates. Furthermore, the results suggest that oxidation of Fe2+ in the presence of lipid or H2O2 creates forms of iron which differ from those formed during simple Fe2+ autoxidation.     

Iron acquisition and virulence in Helicobacter pylori: a major role for FeoB, a high-affinity ferrous iron transporter

The genome sequence of Helicobacter pylori suggests that this bacterium possesses several Fe acquisition systems, including both Fe2+- and Fe3+-citrate transporters. The role of these transporters was investigated by generating insertion mutants in feoB, tonB, fecA1 and fecDE. Fe transport in the feoB mutant was approximately 10-fold lower than in the wild type (with 0.5 microM Fe), irrespective of whether Fe was supplied in the Fe2+ or Fe3+ form. In contrast, transport rates were unaffected by the other mutations. Complementation of the feoB mutation fully restored both Fe2+ and Fe3+ transport. The growth inhibition exhibited by the feoB mutant in Fe-deficient media was relieved by human holo-transferrin, holo-lactoferrin and Fe3+-dicitrate, but not by FeSO4. The feoB mutant had less cellular Fe and was more sensitive to growth inhibition by transition metals in comparison with the wild type. Biphasic kinetics of Fe2+ transport in the wild type suggested the presence of high- and low-affinity uptake systems. The high-affinity system (apparent Ks = 0.54 microM) is absent in the feoB mutant. Transport via FeoB is highly specific for Fe2+ and was inhibited by FCCP, DCCD and vanadate, indicating an active process energized by ATP. Ferrozine inhibition of Fe2+ and Fe3+ uptake implied the concerted involvement of both an Fe3+ reductase and FeoB in the uptake of Fe supplied as Fe3+. Taken together, the results are consistent with FeoB-mediated Fe2+ uptake being a major pathway for H. pylori Fe acquisition. feoB mutants were unable to colonize the gastric mucosa of mice, indicating that FeoB makes an important contribution to Fe acquisition by H. pylori in the low-pH, low-O2 environment of the stomach.     

NADPH- and adriamycin-dependent microsomal release of iron and lipid peroxidation

In a previous study (Minotti, G., 1989, Arch. Biochem. Biophys. 268, 398-403) NADPH-supplemented microsomes were found to reduce adriamycin (ADR) to semiquinone free radical (ADR-.), which in turn autoxidized at the expense of oxygen to regenerate ADR and form O2-. Redox cycling of ADR was paralleled by reductive release of membrane-bound nonheme iron, as evidenced by mobilization of bathophenanthroline-chelatable Fe2+. In the present study, iron release was found to increase with concentration of ADR in a superoxide dismutase- and catalase-insensitive manner. This suggested that membrane-bound iron was reduced by ADR-. with negligible contribution by O2-. or interference by its dismutation product H2O2. Following release from microsomes, Fe2+ was reconverted to Fe3+ via two distinct mechanisms: (i) catalase-inhibitable oxidation by H2O2 and (ii) catalase-insensitive autoxidation at the expense of oxygen, which occurred upon chelation by ADR and increased with the ADR:Fe2+ molar ratio. Malondialdehyde formation, indicative of membrane lipid peroxidation, was observed when approximately 50% of Fe2+ was converted to Fe3+. This occurred in presence of catalase and low concentrations of ADR, which prevented Fe2+ oxidation and favored only partial Fe2+ autoxidation, respectively. Lipid peroxidation was inhibited by superoxide dismutase via increased formation of H2O2 from O2-. and excessive Fe2+ oxidation. Lipid peroxidation was also inhibited by high concentrations of ADR, which favored maximum Fe2+ release but also caused excessive Fe2+ autoxidation via formation of very high ADR:Fe2+ molar ratios. These results highlighted multiple and diverging effects of ADR, O2-., and H2O2 on iron release, iron (auto-)oxidation and lipid peroxidation. Stimulation of malondialdehyde formation by catalase suggested that lipid peroxidation was not promoted by reaction of Fe2+ with H2O2 and formation of hydroxyl radical. The requirement for both Fe2+ and Fe3+ was indicative of initiation by some type of Fe2+/Fe3+ complex.     

Bactericidal effect of Fe2+, ceruloplasmin, and phosphate.

Fe2+, when combined with ceruloplasmin or phosphate, was bactericidal to Escherichia coli at pH 5.0, and when Fe2+, ceruloplasmin, and phosphate were combined, a bactericidal effect was observed under conditions, i.e., short incubation period, in which Fe2+ plus ceruloplasmin and Fe2+ plus phosphate were ineffective. Bactericidal activity increased with the ceruloplasmin or phosphate concentration to a maximum and then decreased as their concentration was further increased. Fe2+ was oxidized in the presence of ceruloplasmin, phosphate, or, in particular, a combination of the two. A bactericidal effect was observed when there was only a partial loss of Fe2+, with more extensive oxidation resulting in a loss of bactericidal activity. The bactericidal effect of Fe2+ plus ceruloplasmin and/or phosphate was unaffected by catalase or superoxide dismutase and was not associated with iodination. Fe-EDTA was also bactericidal at an Fe2+: EDTA molar ratio of 1:0.5, where Fe2+ was partially oxidized. However, in contrast to Fe2+ plus ceruloplasmin and/or phosphate, bactericidal activity was inhibited by catalase and was associated with iodination. Combinations of Fe2+ and Fe3+ were not bactericidal under the conditions employed. A requirement for Fe2+ plus either a product of Fe2+ oxidation or an iron ceruloplasmin and/or phosphate chelate for bactericidal activity is proposed.     

Generation of *OH initiated by interaction of Fe2+ and Cu+ with dioxygen; comparison with the Fenton chemistry

Iron and copper toxicity has been presumed to involve the formation of hydroxyl radical (*OH) from H2O2 in the Fenton reaction. The aim of this study was to verify that Fe2+-O2 and Cu+-O2 chemistry is capable of generating *OH in the quasi physiological environment of Krebs-Henseleit buffer (KH), and to compare the ability of the Fe2+-O2 system and of the Fenton system (Fe2+ + H2O2) to produce *OH. The addition of Fe2+ and Cu+ (0-20 microM) to KH resulted in a concentration-dependent increase in *OH formation, as measured by the salicylate method. While Fe3+ and Cu2+ (0-20 microM) did not result in *OH formation, these ions mediated significant *OH production in the presence of a number of reducing agents. The *OH yield from the reaction mediated by Fe2+ was increased by exogenous Fe3+ and Cu2+ and was prevented by the deoxygenation of the buffer and reduced by superoxide dismutase, catalase, and desferrioxamine. Addition of 1 microM, 5 microM or 10 microM Fe2+ to a range of H2O2 concentrations (the Fenton system) resulted in a H2O2-concentration-dependent rise in *OH formation. For each Fe2+ concentration tested, the *OH yield doubled when the ratio [H2O2]:[Fe2+] was raised from zero to one. In conclusion: (i) Fe2+-O2 and Cu+-O2 chemistry is capable of promoting *OH generation in the environment of oxygenated KH, in the absence of pre-existing superoxide and/or H2O2, and possibly through a mechanism initiated by the metal autoxidation; (ii) The process is enhanced by contaminating Fe3+ and Cu2+; (iii) In the presence of reducing agents also Fe3+ and Cu2+ promote the *OH formation; (iv) Depending on the actual [H2O2]:[Fe2+] ratio, the efficiency of the Fe2+-O2 chemistry to generate *OH is greater than or, at best, equal to that of the Fe2+-driven Fenton reaction.     

Antioxidant properties of S-adenosyl-L-methionine in Fe(2+)-initiated oxidations

S-Adenosylmethionine (SAM) is protective against a variety of toxic agents that promote oxidative stress. One mechanism for this protective effect of SAM is increased synthesis of glutathione. We evaluated whether SAM is protective via possible antioxidant-like activities. Aerobic Hepes-buffered solutions of Fe2+ spontaneously oxidize and consume O2 with concomitant production of reactive oxygen species and oxidation of substrates to radical products, e.g., ethanol to hydroxyethyl radical. SAM inhibited this oxidation of ethanol and inhibited aerobic Fe2+ oxidation and consumption of O2. SAM did not regenerate Fe2+ from Fe3+ and was not consumed after incubation with Fe2+. SAM less effectively inhibited aerobic Fe2+ oxidation in the presence of competing chelating agents such as EDTA, citrate, and ADP. The effects of SAM were mimicked by S-adenosylhomocysteine, but not by methionine or methylthioadenosine. SAM did not inhibit Fe2+ oxidation by H2O2 and was a relatively poor inhibitor of the Fenton reaction. Lipid peroxidation initiated by Fe2+ in liposomes was associated with Fe2+ oxidation; these two processes were inhibited by SAM. However, SAM did not show significant peroxyl radical scavenging activity. SAM also inhibited the nonenzymatic lipid peroxidation initiated by Fe2+ + ascorbate in rat liver microsomes. These results suggest that SAM inhibits alcohol and lipid oxidation mainly by Fe2+ chelation and inhibition of Fe2+ autoxidation. This could represent an important mechanism by which SAM exerts cellular protective actions and reduces oxidative stress in biological systems.     

Ferritin-catalyzed consumption of hydrogen peroxide by amine buffers causes the variable Fe2+ to O2 stoichiometry of iron deposition in horse spleen ferritin

Ferritin catalyzes the oxidation of Fe2+ by O2 to form a reconstituted Fe3+ oxy-hydroxide mineral core, but extensive studies have shown that the Fe2+ to O2 stoichiometry changes with experimental conditions. At Fe2+ to horse spleen ferritin (HoSF) ratios greater than 200, an upper limit of Fe2+ to O2 of 4 is typically measured, indicating O2 is reduced to 2H2O. In contrast, a lower limit of Fe2+ to O2 of approximately 2 is measured at low Fe2+ to HoSF ratios, implicating H2O2 as a product of Fe2+ deposition. Stoichiometric amounts of H2O2 have not been measured, and H2O2 is proposed to react with an unknown system component. Evidence is presented that identifies this component as amine buffers, including 3-N-morpholinopropanesulfonic acid (MOPS), which is widely used in ferritin studies. In the presence of non-amine buffers, the Fe2+ to O2 stoichiometry was approximately 4.0, but at high concentrations of amine buffers (0.10 M) the Fe2+ to O2 stoichiometry is approximately 2.5 for iron loadings of eight to 30 Fe2+ per HoSF. Decreasing the concentration of amine buffer to zero resulted in an Fe2+ to O2 stoichiometry of approximately 4. Direct evidence for amine buffer modification during Fe2+ deposition was obtained by comparing authentic and modified buffers using mass spectrometry, NMR, and thin layer chromatography. Tris(hydroxymethyl)aminomethane, MOPS, and N-methylmorpholine (a MOPS analog) were all rapidly chemically modified during Fe2+ deposition to form N-oxides. Under identical conditions no modification was detected when amine buffer, H2O2, and O2 were combined with Fe2+ or ferritin separately. Thus, a short-lived ferritin intermediate is required for buffer modification by H2O2. Variation of the Fe2+ to O2 stoichiometry versus the Fe2+ to HoSF ratio and the amine buffer concentration are consistent with buffer modification.     

Stimulatory effect of ferrous ion on mildiomycin production by Streptoverticillium rimofaciens

The minimal concentration of Fe 2+ necessary for the maximal production of mildiomycin by Streptoverticillium rimo-faciens was about 8 mg Fe 2+ /ml at which the production of mildiomycin was about 10 times higher than that in the culture without added Fe 2+ . The culture with added Fe 2+ had a marked increase in NH 4 + assimilation, followed by an increase in mildiomycin production. The ferrous ion was incorporated during the cell growth and the uptake capacity of Fe 2+ was changed according to inoculum age and associated with the productivity of mildiomycin.     

Direct Measurement of 59Fe-Labeled Fe2+ Influx in Roots of Pea Using a Chelator Buffer System to Control Free Fe2+ in Solution

Fe2+ transport in plants has been difficult to quantify because of the inability to control Fe2+ activity in aerated solutions and non-specific binding of Fe to cell walls. In this study, a Fe(II)-3-(2-pyridyl)-5,6-diphenyl-1,2,4-triazine-4[prime]4"-disulfonic acid buffer system was used to control free Fe2+ in uptake solutions. Additionally, desorption methodologies were developed to adequately remove nonspecifically bound Fe from the root apoplasm. This enabled us to quantify unidirectional Fe2+ influx via radiotracer (59Fe) uptake in roots of pea (Pisum sativum cv Sparkle) and its single gene mutant brz, an Fe hyperaccumulator. Fe influx into roots was dramatically inhibited by low temperature, indicating that the measured Fe accumulation in these roots was due to true influx across the plasma membrane rather than nonspecific binding to the root apoplasm. Both Fe2+ influx and Fe translocation to the shoots were stimulated by Fe deficiency in Sparkle. Additionally, brz, a mutant that constitutively exhibits high ferric reductase activity, exhibited higher Fe2+ influx rates than +Fe-grown Sparkle. These results suggest that either Fe deficiency triggers the induction of the Fe2+ transporter or that the enhanced ferric reductase activity somehow stimulates the activity of the existing Fe2+ transport protein.     

Physicochemical characterization of the microbial Fe2+ chelator proferrorosamine from Pseudomonas roseus fluorescens.

The microbial chelating compound proferrorosamine A, produced by Pseudomonas roseus fluorescens, formed a complex with Fe2+ of which the apparent stability constant was found to be 10(23). The following order of increasing stability constants of metal complexes with proferrorosamine was established as: Ba2+, Ca2+, Mg2+, Mn2+ less than Hg2+ less than Zn2+ less than Pb2+ less than Co2+ less than Cu2+ congruent to Fe2+ less than Ni2+. Only Ni(2+)-proferrorosamine had a stability constant which was established as: Ba2+, Ca2+, Mg2+, Mn2+ less than Hg2+ less than Zn2+ less than Pb2+ less than Co2+ less than Cu2+ congruent to Fe2+ less than Ni2+. Only Ni(2+)-proferrorosamine had a stability constant which was ca 32 times higher than Fe(2+)-proferrorosamine. Because of the production of proferrorosamine the growth of Ps. roseus fluorescens was not inhibited in iron limiting media by the addition of 0.15 mmol/l of the weaker chemical Fe2+ chelator 2,2'-dipyridyl. This contrasted with the proferrorosamine-negative mutant K2 and Ps. stutzeri, which only produces Fe(3+)-chelating siderophores. Furthermore, it was found that proferrorosamine was able to dissolve Fe2+ from stainless steel. These results show that proferrorosamine is a strong and selective Fe2+ chelator which could be used as an alternative for the toxic 2,2'-dipyridyl to control lactic acid fermentations.     

The CorA Mg2+ transporter does not transport Fe2+

corA encodes the constitutively expressed primary Mg2+ uptake system of most eubacteria and many archaea. Recently, a mutation in corA was reported to make Salmonella enterica serovar Typhimurium markedly resistant to Fe2+-mediated toxicity. Mechanistically, this was hypothesized to be from an ability of CorA to mediate the influx of Fe2+. Consequently, we directly examined Fe2+ transport and toxicity in wild-type versus corA cells. As determined by direct transport assay, CorA cannot transport Fe2+ and Fe2+ does not potently inhibit CorA transport of 63Ni2+. Mg2+ can, relatively weakly, inhibit Fe2+ uptake, but inhibition is not dependent on the presence of a functional corA allele. Although excess Fe2+ was slightly toxic to S. enterica serovar Typhimurium, we were unable to elicit a significant differential sensitivity in a wild-type versus a corA strain. We conclude that CorA does not transport Fe2+ and that the relationship, if any, between iron toxicity and corA is indirect.     

Microsomal reduction of low-molecular-weight Fe3+ chelates and ferritin: enhancement by adriamycin, paraquat, menadione, and anthraquinone 2-sulfonate and inhibition by oxygen

Reduction of iron is important in promoting xenobiotic-enhanced, microsomal lipid peroxidation, yet there is little evidence that Fe3+ chelates that promote lipid peroxidation can be reduced by the microsomal system. We have shown that rat liver microsomes catalyse NADPH-dependent reduction of Fe3+ without chelator, as well as Fe3+(ADP), Fe3+(ATP), Fe3+(citrate), Fe3+(EDTA), and ferrioxamine in N2. The NADPH oxidation that accompanied Fe3+ reduction was inhibited by CO for all chelates, except Fe3+ (EDTA). This implies that, except for Fe3+ (EDTA), cytochrome P450 was involved in reduction of the complexes. Adriamycin, paraquat, and anthraquinone 2-sulfonate (AQS) enhanced reduction of all the Fe3+ chelates, whereas menadione enhanced reduction only of Fe3+(ADP) and Fe3+(citrate). All the compounds enhanced oxidation of NADPH in the presence or absence of iron. This was not inhibited by CO, and the results are compatible with Fe3+ reduction occurring via the xenobiotic radicals produced by cytochrome P450 reductase. Microsomal reduction of the xenobiotics, except menadione, enabled the reduction and release of iron from ferritin. Fe3+ chelate reduction, both with and without xenobiotic, was inhibited by O2, although it still proceeded in air at 10-20% of the rate in N2. Iron-dependent lipid peroxidation was promoted by ADP and ATP, inhibited 50% by citrate, and completely inhibited by EDTA and desferrioxamine. Of the xenobiotics, only Adriamycin enhanced microsomal lipid peroxidation. These results indicate that the effects of chelators and xenobiotics on Fe3+ reduction do not correlate with lipid peroxidation and, although reduction is necessary, there must be other factors involved.     

Lipid peroxidation and cytotoxicity of Ehrlich ascites tumor cells by ferric nitrilotriacetate

Ferric nitrilotriacetate, which causes in vivo organ injury, induced lipid peroxidation and cell death in Ehrlich ascites tumor cells in vitro. The process was inhibited by butylated hydroxyanisole and enhanced by vitamin C and linolenic acid, indicating a close relationship between cytotoxicity and the lipid peroxidizing ability of Fe3+ NTA. The cytotoxicity was suppressed by glucose and a temperature below 20 degrees C. Lipid peroxidation of Fe3+ NTA-treated cells was greater at 0 degree C than at 37 degrees C, contrary to results with Fe3+ NTA-treated plasma membranes of Ehrlich ascites tumor cell. These results suggested that metabolism and membrane fluidity are important factors in the expression of the Fe3+ NTA-induced cytotoxicity. H2O2 showed a lower cytotoxicity than did Fe3+ NTA but a greater lipid peroxidizing ability. H2O2 appeared to damage the cells less, and was quenched rapidly by cellular metabolism unlike Fe3+ NTA. In transferrin-free medium, Ehrlich ascites tumor cell readily incorporated Fe3+ NTA, and iron uptake was greater than NTA-uptake in Fe3+ NTA-treated cells, suggesting that Ehrlich ascites tumor cell incorporated iron from Fe3+NTA and metabolized it into an inert form such as ferritin.     

Factors affecting the manganese and iron activation of the phosphoenolpyruvate carboxykinase isozymes from rabbit.

Timed assays in which GTP and GDP were separated and quantitated by HPLC were developed and used to study the metal activation of the mitochondrial and cytosolic isozymes of phosphoenolpyruvate carboxykinase purified from rabbit liver. These assays allowed both directions of catalysis to be studied under similar conditions and in the absence of coupling enzymes. The mitochondrial enzyme is rapidly inactivated by preincubation with Fe2+, as had been shown previously for the cytosolic isozyme. The greatest activation by Fe2+ was obtained by adding micromolar Fe2+ immediately after enzyme to form the complete assay mixture that also contained millimolar Mg2+. In the direction of synthesis of OAA from Pep, the K0.5 values for Mn2+ and Fe2+ were in the 3-7 microM range when a nonchelating buffer, Hepes, was used. The buffer used strongly affected activation by Fe2+ at pH 7.4; activation was eliminated in the case of phosphate and K0.5 increased several-fold over that obtained with Hepes when imidazole was used. In non-chelating buffer, the pH optimum was near 7.4 for both isozymes and for both directions of catalysis. However, the near optimal pH range extended below 7.4 for the direction of oxaloacetate synthesis while the range was above 7.4 for Pep synthesis. In the direction of oxaloacetate synthesis: (1) Both isozymes required the presence of micromolar Mn2+ or Fe2+ in addition to millimolar Mg2+ in order to shown significant activity. (2) Fe2+ was as effective an activator as Mn2+ at pH 7 and below. In the direction of Pep synthesis: (1) Micromolar Mn2+ was a much better activator than Fe2+ at the higher pH values needed for optimal activity in this direction. (2) With increasing pH, decreasing activation was obtained with Fe2+ while the activity supported by Mg2+ alone increased. The results demonstrate the potential for regulation of either isozyme of Pep carboxykinase by the availability of iron or manganese.     

Spin trapping study on the kinetics of Fe2+ autoxidation: formation of spin adducts and their destruction by superoxide.

The oxidation of Fe2+ was investigated by electron spin resonance spin trapping techniques with N-t-butyl-alpha-phenylnitrone (PBN) and dimethyl sulfoxide. Under pure oxygen, the spin adduct PBN/.OCH3 was rapidly generated by the addition of Fe2+ (0.2-1.2 mM) into phosphate buffer containing ethylenediaminetetraacetate (EDTA), dimethyl sulfoxide, and PBN at pH 7.4, but it decayed. The decay process of PBN/.OCH3 consists of two components. The fast decay was dependent on Fe2+ concentration. Another was due to destruction of the spin adduct by superoxide anion (.O2-), because superoxide dismutase (SOD) markedly prevented the decay. Catalase decreased the yield of PBN/.OCH3. When EDTA was replaced by diethylenetriaminepentaacetic acid (DTPA), both the generation and decay process of PBN/.OCH3 were slow. SOD and catalase effects were similar to those in EDTA. Fe2+ produced PBN/.OCH3 even in the absence of chelators. We could estimate the kinetic parameters by computer simulation, comparing the Fe2+ oxidation in EDTA with that in DTPA. These results demonstrate that Fe2+ reacts with O2 to generate .O2- and then H2O2, which produces .CH3 by reaction with Fe2+ and dimethyl sulfoxide.(.)OCH3 results from the reaction between .CH3 and O2. The adduct PBN/.OCH3 decays by reaction with Fe2+ and .O2-.     

Effect of Ca2+ and Mg2+ on the uptake of Fe3+ by mouse intestinal mucosa

Initial Fe3+ uptake rates by mouse intestinal fragments were determined in vitro. Uptake was dependent primarily on the Fe3+-nitrilotriacetate complex concentration. Addition of Ca2+ and Mg2+ to the incubation medium had only small effects on the Fe3+ uptake rate. Duodenal fragments from hypoxic animals showed enhanced uptake of Fe3+; this increase was more pronounced with a divalent cation-free medium. Ca2+ markedly diminished the Fe3+ uptake by mucosa from hypoxic mice; Mg2+ had no appreciable effect. Distal ileal fragments exhibited lower uptake rates compared to the duodenum, but were more sensitive to the effects of added Ca2+. The ileal fragments did not show an adaptive response of Fe3+ uptake to hypoxia. These results suggest the existence of more than one pathway for mucosal Fe3+ uptake. One pathway, sensitive to Ca2+ and not stimulated by hypoxia, may be present in the duodenum and ileum. A second pathway, inhibited by Ca2+ and exhibiting an adaptive response to hypoxia, occurs only in the duodenum. This latter pathway is more sensitive to the effects of metabolic inhibitors.     

金属离子对黑米花青苷色素吸收光谱的影响

以黑糯B糙米皮为实验材料 ,用 1 .5mol/L盐酸— 95 %乙醇 (V/V :1 5 / 85 )溶液提取黑米花青苷色素(BRAP) ,采用紫外可见分光光度法研究了 1 1种金属离子以及 (NH4 ) 1+ 离子对BRAP的作用。结果表明 ,未加离子条件下色素溶液可见光区λmax5 35nm ,紫外光区λmax2 80nm ,加入Al3 + 、Fe3 + 、Fe2 + 、Cu2 + 、Mn2 + 、Zn2 + 、Sn2 + 对其吸收光谱有显性影响。其中Al3 + 、Fe3 + 使 5 35nm特征吸收峰发生蓝移 ,Sn2 + 使其发生明显红移 ;Al3 + ,Fe2 + ,Mn2 + ,Zn2 +在 5 35nm附近有增加ABS值作用 ,Fe3 + 有减小ABS值作用 ;延长作用时间 ,Cu2 + 对BRAP吸收光谱的影响表现为λmax5 35nm发生蓝移 ,ABS值减小     

Characterization of tissue tolerance to iron by molecular markers in different lines of rice

Ferrous iron (Fe2+) toxicity is a major disorder in rice prod uction on acid, flooded soils. Rice ( Oryza sativa L.) genotypes differ widely in tolerance to Fe2+ toxicity, which makes it possible to bre ed more tolerant rice varieties. Tissue tolerance to higher iron concentrations in plants has been considered to be important to Fe2+ tolerance in ri ce. Segregation for leaf bronzing and growth reduction due to Fe2+ to xicity was observed in a doubled haploid (DH) population with 135 lines derived from a Fe2+ tolerant japonica variety, Azucena, and a sensitive indic a variety, IR64 in a solution culture with Fe2+ stress condition at a Fe2+concentration of 250 mg L-1 at pH 4.5. To better understand the mechanism of tissue tolerance, Leaf Bronzing Index (LBI), total iron concentration in shoot tissue and the enzymes of ascorbate peroxidase (AP), dehydroascorbate reductase (DR) and glutathione reductase (GR), and concentrations of ascorbate (AS) and dehydroascorbate (DHA), which are involved in the ascorbate-specific H2O2-scavenging system, were determined for the population under Fe2+ stress. A non-normal distribution of LBI was found. About 38 lines showed no bronzing, while the lines with non-zero LBI values ranged from 0.05 to 0.85 and showed a normal distribution. The other parameters measured showed normal distribution. The total iron concentrations in the 38 tolerant lines ranged from 1.76 mg Fe g-1 to 4.12 mg Fe g-1 and was in a similar range as in the non-tolerant genotype (2.04 – 4.55 mg Fe g-1). No significant differences in the activities of the enzymes were found between the parents under normal culture, but remarkably higher Fe2+ induced enzyme activities were observed in the tolerant parent. AS was similar between the parents under both normal and Fe2+ stress, but its concentration was sharply decreased under Fe2+ stress. DHA was much lower in the tolerant parent than in the sensitive parent under Fe2+ stress. Single locus analysis and interval mapping analysis based on 175 molecular markers revealed that the interval flanked by RG345 and RZ19 on chromosome one was an important location of gene(s) for Fe2+ tolerance. The ascorbate-specific system for scavenging Fe2+-mediated oxygen free radicals may be an important mechanism for tissue Fe2+ tolerance. A gene locus with relative small effect on root ability to exclude Fe2+ was also detected.