The accessibility of iron at the active site of recombinant human phenylalanine hydroxylase to water as studied by 1H NMR paramagnetic relaxation. Effect of L-Phe and comparison with the rat enzyme.
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The high-spin (S = 5/2) Fe(III) ion at the active site of recombinant human phenylalanine hydroxylase (PAH) has a paramagnetic effect on the longitudinal relaxation rate of water protons. This effect is proportional to the concentration of enzyme, with a paramagnetic molar-relaxivity value at 400 MHz and 25 degrees C of 1. 3 (+/- 0.03) x 10(3) s-1 M-1. The value of the Arrhenius activation energy (Ea) for the relaxation rate was -14.4 +/- 1.1 kJ/mol for the resting enzyme, indicating a fast exchange of water protons in the paramagnetic environment. The frequency dependence of the relaxation rate also supported this hypothesis. Thus, the recombinant human PAH appears to have a more solvent-accessible catalytic iron than the rat enzyme, in which the water coordinated to the metal is slowly exchanging with the solvent. These findings may be related to the level of basal activity before activation for these enzymes, which is higher for human than for rat PAH. In the presence of saturating (5 mM) concentrations of the substrate L-Phe, the paramagnetic molar relaxivity for human PAH decreased to 0.72 (+/- 0.05) x 10(3) s-1 M-1 with no significant change in the Ea. Effective correlation times (tauC) of 1.8 (+/- 0.3) x 10(-10) and 1.25 (+/- 0.2) x 10(-10) s-1 were calculated for the enzyme and the enzyme-substrate complex, respectively, and most likely represent the electron spin relaxation rate (tauS) for Fe(III) in each case. Together with the paramagnetic molar-relaxivity values, the tauC values were used to estimate Fe(III)-water distances. It seems that at least one of the three water molecules coordinated to the iron in the resting rat and human enzymes is displaced from coordination on the binding of L-Phe at the active site. (+info)
Ferritin mutants of Escherichia coli are iron deficient and growth impaired, and fur mutants are iron deficient.
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Escherichia coli contains at least two iron storage proteins, a ferritin (FtnA) and a bacterioferritin (Bfr). To investigate their specific functions, the corresponding genes (ftnA and bfr) were inactivated by replacing the chromosomal ftnA and bfr genes with disrupted derivatives containing antibiotic resistance cassettes in place of internal segments of the corresponding coding regions. Single mutants (ftnA::spc and bfr::kan) and a double mutant (ftnA::spc bfr::kan) were generated and confirmed by Western and Southern blot analyses. The iron contents of the parental strain (W3110) and the bfr mutant increased by 1.5- to 2-fold during the transition from logarithmic to stationary phase in iron-rich media, whereas the iron contents of the ftnA and ftnA bfr mutants remained unchanged. The ftnA and ftnA bfr mutants were growth impaired in iron-deficient media, but this was apparent only after the mutant and parental strains had been precultured in iron-rich media. Surprisingly, ferric iron uptake regulation (fur) mutants also had very low iron contents (2.5-fold less iron than Fur+ strains) despite constitutive expression of the iron acquisition systems. The iron deficiencies of the ftnA and fur mutants were confirmed by Mossbauer spectroscopy, which further showed that the low iron contents of ftnA mutants are due to a lack of magnetically ordered ferric iron clusters likely to correspond to FtnA iron cores. In combination with the fur mutation, ftnA and bfr mutations produced an enhanced sensitivity to hydroperoxides, presumably due to an increase in production of "reactive ferrous iron." It is concluded that FtnA acts as an iron store accommodating up to 50% of the cellular iron during postexponential growth in iron-rich media and providing a source of iron that partially compensates for iron deficiency during iron-restricted growth. In addition to repressing the iron acquisition systems, Fur appears to regulate the demand for iron, probably by controlling the expression of iron-containing proteins. The role of Bfr remains unclear. (+info)
Identification and characterization of SirA, an iron-regulated protein from Staphylococcus aureus.
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The acquisition of iron by pathogenic bacteria is often a crucial step in establishing infection. To accomplish this, many bacteria, including Staphylococcus aureus, produce low-molecular-weight iron-chelating siderophores. However, the secretion and transport of these molecules in gram-positive organisms are poorly understood. The sequence, organization, and regulation of genes involved in siderophore transport are conserved among gram-negative bacteria. We used this information to identify a putative siderophore transport locus from an S. aureus genomic sequence database. This locus contains three predicted open reading frames with a high degree of homology to genes involved in siderophore uptake in several bacterial species, in particular the cbr locus of the plant pathogen Erwinia chrysanthemi. The first gene in the locus, which we have designated sir for staphylococcal iron regulated, encodes a putative lipoprotein with a molecular mass of 37 kDa. The open reading frame is preceded by a 19-bp region of dyad symmetry with homology for operator sequences controlling iron-regulated expression of genes in other bacteria. Fur titration experiments indicate that this region of dyad symmetry is sufficient for Fur-dependent regulation in Escherichia coli. The expression of this gene was repressed, in a dose-dependent manner, by the addition of iron to the S. aureus culture medium. sir-encoded proteins may be involved in iron acquisition in vivo and therefore may be targets for antimicrobial agents. (+info)
The iron transport protein NRAMP2 is an integral membrane glycoprotein that colocalizes with transferrin in recycling endosomes.
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The natural resistance associated macrophage protein (Nramp) gene family is composed of two members in mammals, Nramp1 and Nramp2. Nramp1 is expressed primarily in macrophages and mutations at this locus cause susceptibility to infectious diseases. Nramp2 has a much broader range of tissue expression and mutations at Nramp2 result in iron deficiency, indicating a role for Nramp2 in iron metabolism. To get further insight into the function and mechanism of action of Nramp proteins, we have generated isoform specific anti-Nramp1 and anti-Nramp2 antisera. Immunoblotting experiments indicate that Nramp2 is present in a number of cell types, including hemopoietic precursors, and is coexpressed with Nramp1 in primary macrophages and macrophage cell lines. Nramp2 is expressed as a 90-100-kD integral membrane protein extensively modified by glycosylation (>40% of molecular mass). Subcellular localization studies by immunofluorescence and confocal microscopy indicate distinct and nonoverlapping localization for Nramp1 and Nramp2. Nramp1 is expressed in the lysosomal compartment, whereas Nramp2 is not detectable in the lysosomes but is expressed primarily in recycling endosomes and also, to a lower extent, at the plasma membrane, colocalizing with transferrin. These findings suggest that Nramp2 plays a key role in the metabolism of transferrin-bound iron by transporting free Fe2+ across the endosomal membrane and into the cytoplasm. (+info)
Effect of meat (beef, chicken, and bacon) on rat colon carcinogenesis.
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High intake of red meat or processed meat is associated with increased risk of colon cancer. In contrast, consumption of white meat (chicken) is not associated with risk and might even reduce the occurrence of colorectal cancer. We speculated that a diet containing beef or bacon would increase and a diet containing chicken would decrease colon carcinogenesis in rats. One hundred female Fischer 344 rats were given a single injection of azoxymethane (20 mg/kg i.p.), then randomized to 10 different AIN-76-based diets. Five diets were adjusted to 14% fat and 23% protein and five other diets to 28% fat and 40% protein. Fat and protein were supplied by 1) lard and casein, 2) olive oil and casein, 3) beef, 4) chicken with skin, and 5) bacon. Meat diets contained 30% or 60% freeze-dried fried meat. The diets were given ad libitum for 100 days, then colon tumor promotion was assessed by the multiplicity of aberrant crypt foci [number of crypts per aberrant crypt focus (ACF)]. The ACF multiplicity was nearly the same in all groups, except bacon-fed rats, with no effect of fat and protein level or source (p = 0.7 between 8 groups by analysis of variance). In contrast, compared with lard- and casein-fed controls, the ACF multiplicity was reduced by 12% in rats fed a diet with 30% bacon and by 20% in rats fed a diet with 60% bacon (p < 0.001). The water intake was higher in bacon-fed rats than in controls (p < 0.0001). The concentrations of iron and bile acids in fecal water and total fatty acids in feces changed with diet, but there was no correlation between these concentrations and the ACF multiplicity. Thus the hypothesis that colonic iron, bile acids, or total fatty acids can promote colon tumors is not supported by this study. The results suggest that, in rats, beef does not promote the growth of ACF and chicken does not protect against colon carcinogenesis. A bacon-based diet appears to protect against carcinogenesis, perhaps because bacon contains 5% NaCl and increased the rats' water intake. (+info)
Ferroxidase activity of ferritin: effects of pH, buffer and Fe(II) and Fe(III) concentrations on Fe(II) autoxidation and ferroxidation.
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It is widely accepted that iron deposition in the iron storage protein ferritin in vitro involves Fe(II) oxidation, and that ferritin facilitates this oxidation at a ferroxidase site on the protein. However, these views have recently been questioned, with the protein ferroxidase activity instead being attributed to autoxidation from the buffer alone. Ligand exchange between another protein with ferroxidase activity and ferritin has been proposed as an alternative mechanism for iron incorporation into ferritin. In the present work, a pH stat apparatus is used to eliminate the influence of buffers on iron(II) oxidation. Here we show that the recent experiments questioning the ferroxidase activity of ferritin were flawed by inadequate pH control, that buffers actually retard rather than facilitate iron(II) oxidation, and that horse spleen ferritin has ferroxidase activity when measured under proper experimental conditions. Furthermore, high pH (7.0), a high Fe(II) concentration and the presence of Fe(III) all favour Fe(II) autoxidation in the presence or absence of ferritin. (+info)
Quantitative study of the variability of hepatic iron concentrations.
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BACKGROUND: The hepatic iron concentration (HIC) is widely used in clinical practice and in research; however, data on the variability of HIC among biopsy sites are limited. One aim of the present study was to determine the variability of HIC within both healthy and cirrhotic livers. METHODS: Using colorimetric methods, we determined HIC in multiple large (microtome) and small (biopsy-sized) paraffin-embedded samples in 11 resected livers with end-stage cirrhosis. HIC was also measured in multiple fresh samples taken within 5 mm of each other ("local" samples) and taken at sites 3-5 cm apart ("remote" samples) from six livers with end-stage cirrhosis and two healthy autopsy livers. RESULTS: The within-organ SD of HIC was 13-1553 microg/g (CV, 3.6-55%) for microtome samples and 60-2851 microg/g (CV, 15-73%) for biopsy-sized samples. High variability of HIC was associated with mild to moderate iron overload, because the HIC SD increased with increasing mean HIC (P <0.002). Livers with mean HIC >1000 microg/g exhibited significant biological variability in HIC between sites separated by 3-5 cm (remote sites; P <0.05). The SD was larger for biopsy-sized samples than for microtome samples (P = 0.02). CONCLUSION: Ideally, multiple hepatic sites would be sampled to obtain a representative mean HIC. (+info)
Separate and joint effects of micronutrient deficiencies on linear growth.
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Recent studies have investigated the effect of micronutrient deficiencies on growth stunting, with special attention toward the effect of zinc, iron, vitamin A and iodine deficiencies. In Mexico, the prevalence of growth stunting in children <5 y old is approximately 24%; it is higher in rural areas and lower in urban areas. In an initial study, the effect of zinc and/or iron supplementation on linear growth was investigated in a longitudinal, placebo-controlled design. After 12 mo of supplementation, there was no difference between the groups supplemented with zinc, iron or zinc plus iron and the placebo group. At baseline, 82% of the children in this study were deficient in at least two out of the five micronutrients that were determined, and 73% were anemic. In another study, a mixture of those micronutrients that were documented to be lacking in Mexican children was formulated in a supplement and given to Mexican children over a period of 12 mo in a longitudinal, placebo-controlled, supplementation design. Children in the low and medium socioeconomic status grew about 1 cm more than similar children in the placebo group. This difference was not found in children of high socioeconomic status. It is suggested that, in most cases, growth stunting is associated with marginal deficiencies of several micronutrients and that in populations with multiple micronutrient deficiencies, the effect on linear growth of supplementation with single nutrients will not be significant. Supplementation with multiple micronutrients is expected to be more effective, but even in that case the actual increment in height was less than the expected potential increment. (+info)