©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Ascorbic Acid Enhances Ferritin mRNA Translation by an IRP/Aconitase Switch (*)

(Received for publication, March 8, 1995; and in revised form, June 5, 1995)

Ildiko Toth Kenneth R. Bridges (§)

From the Division of Hematology-Oncology, Department of Medicine, Brigham and Women's Hospital, Harvard Medical School, Boston, Massachusetts 02115

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

Replenishment of ascorbate in cultured cells, which are almost uniformly vitamin-deficient, increases ferritin mRNA translation in response to iron by 20-fold (Toth, I., Rogers, J. T., McPhee, J. A., Elliott, S. M., Abramson, S. L., and Bridges, K. R.(1995) J. Biol. Chem. 270, 2846-2852). We now demonstrate that ascorbate increases cytosolic aconitase activity. The iron-responsive element-binding protein (IRP-1) exists in three states: bound to mRNA without aconitase activity, free in the cytosol without aconitase activity, and free in the cytosol with aconitase activity. Ascorbate converts free IRP-1 to the enzymatically active form. Enhanced ferritin synthesis with subsequent iron stimulation is due to the altered equilibrium of the free IRP-1. The cellular biology of iron is closely intertwined with that of ascorbate.


INTRODUCTION

Ascorbic acid, or vitamin C, is a dietary requirement for primates, guinea pigs, and a few other species that fail to synthesize the compound (Szent-Györgyi, 1928; Burns, 1957; Nishikimi and Yagi, 1991). Additionally, most cells in culture are ascorbate-deficient since the vitamin generally is not added to culture media and is extremely labile in solution (Moser and Bendich, 1991). We have shown previously that ascorbic acid retards lysosomal autophagy of cytosolic ferritin (Bridges and Hoffman, 1986; Bridges, 1987; Hoffman et al., 1991).

More recent work in our laboratory demonstrated that ascorbic acid greatly increases the ferritin content of cells subsequently stimulated with iron (Toth et al., 1995). Base-line ferritin synthesis in ascorbate replete or deficient cells is identical. Ferritin message translation in response to iron loading was greater in cells grown in media containing physiological concentrations of ascorbate relative to those grown under standard culture conditions. Ascorbate's role as a facilitator of ferritin synthesis in cells is mediated through the 5`-untranslated region region of the ferritin message containing the iron-responsive element (IRE) (^1)(Rouault et al., 1988; Hentze et al., 1988; Harrell et al., 1991; Kühn and Hentze, 1992). We have extended our investigations and examined ascorbate's effects on IRP-1, a dual function protein that binds the IRE sequence and has aconitase activity (Constable et al., 1992; Philipot etal., 1993; Hirling et al., 1994). Our data indicate that ascorbate transduces enzymatically inactive free IRP-1 to an active form.


EXPERIMENTAL PROCEDURES

Cell Culture

K562 human leukemia cells were maintained in RPMI 1640 medium supplemented with 10% heat-inactivated newborn calf serum and L-glutamine. Log phase growth was assured by maintaining a density of 2-5 10^5 cells/ml.

Metabolic Labeling and Immunoprecipitation of de Novo Synthesized Ferritin

A total of 5 10^6 to 10^7 K562 cells were treated with varying combinations of 10 µg/ml ferric ammonium citrate, 150 mM ascorbic acid, 100 mM desferrioxamine, 500 µM uric acid, or glutathione as outlined for the individual experiments. They were washed twice with PBS buffer and incubated for 1 h in methionine-free RPMI 1640 medium supplemented with [S]methionine at a concentration of 25 µCi/ml. Cells were washed with PBS and lysed with buffer containing 1% Triton X-100, 0.15 M NaCl, 0.02 M Tris-HCl, pH 7.5, and 0.2 mM phenylmethylsulfonyl fluoride (lysis buffer). Newly synthesized ferritin subunits were immunoprecipitated for 1 h with rabbit antibody to human ferritin (Boehringer Mannheim). The antigen-antibody complex was immobilized with protein A-Sepharose and washed extensively. The beads were boiled for 10 min in an SDS phosphate-urea electrophoresis buffer and separated at 80 V constant voltage for 16 h on a 15% polyacrylamide gel containing 6 M urea. After fixation, the gel was treated with autoradiography image enhancer, dried, and used to expose x-ray film for 2-4 days. The intensity of the H- and L-ferritin subunits was quantified with Abaton Scan equipment.

Preparation of Cytosolic Cell Extract from K562 Cells

Cells were harvested and washed twice with PBS buffer and counted. A total of 10^7 cells were lysed in 500 ml of 10 mM HEPES, pH 7.5, 40 mM KCl, 5% glycerol, 3 mM NaCl, 0.3% Nonidet P-40, 1 µM phenylmethylsulfonyl fluoride, 10 µg/ml leupeptin. The extract was centrifuged at 14,000 g for 20 min, followed by 100,000 g for 30 min in a Beckman L8-70M ultracentrifuge. Mitochondrial contamination of the supernatant was excluded by the absence of cytochrome c oxidase activity.

Cytochrome c Oxidase Assay

Cytochrome c oxidase activity was measured according to the method of Smith(1955) (Table 1).



Protein Determination

The protein concentration of the cellular extracts was determined by Bio-Rad micro-assay according to the supplier's manual (Bio-Rad).

Cytosolic Aconitase Activity

Cytosolic aconitase activity was determined by measuring the decrease of cis-aconitate as assessed by spectrophotometric absorbance at 240 nm. Mitochondrial-free K562 lysates (50-100 µg) were incubated with 200 mMcis-aconitate in 50 mM Tris-HCl buffer, pH 7.2, 100 mM NaCl, 0.02% bovine serum albumin at room temperature in a final volume of 1 ml. Specific activity (mmol of substrate converted/mg of protein/min at room temperature) was calculated (Emery-Goodman et al., 1993). To assess the effect of in vitro addition of ascorbate, freshly prepared cell lysate containing cytosolic aconitase was pretreated for 10 min with a fresh solution of ascorbic acid or dehydroascorbic acid at final concentrations ranging between 1 pM and 500 mM, oxidized (GSSG) or reduced glutathione (GSH), or uric acid (stock solution in 100 mM HEPES buffer, pH 7.2) at final concentrations ranging between 1 pM and 1 mM prior to enzyme assay.

Immunoblot Analysis

Proteins were separated by SDS-PAGE on a 10% gel, transferred and immobilized on a nitrocellulose membrane, and hybridized with rabbit antibody raised against human IRP-1 (a generous gift of Dr. Lucas Kühn; Henderson et al., 1993) diluted 1:1000. We found, as reported previously, that the antibody recognized only IRP-1. Horseradish peroxidase-conjugated goat antibody to rabbit IgG (Bio-Rad) was diluted 1:1000 and reacted against the antibody to IRP-1. The position of the antigen-antibody complex was detected using the color enzymatic reaction according to the supplier's manual.

Gel Retardation Assay

IRE-protein interactions were assessed as described (Liebold and Munro, 1988; Müllner et al., 1989). An excess amount of [P]UTP-labeled RNA transcript (from pSPT-fer, a 28-nucleotide oligonucleotide encoding the human H-ferritin IRE; a generous gift of Dr. Kühn; see Müllner et al.(1989)) was incubated with 2 µg of cytoplasmic cell lysate (prepared with lysis buffer as detailed above) at room temperature for 20 min. RNase T1 (1 unit/ml) and heparin (5 mg/ml) were added sequentially for 10 min each. IRE-protein complexes were resolved on a 6% nondenaturing polyacrylamide gel as detailed by Leibold and Munro.


RESULTS

Effect of Ascorbic Acid on Cytosolic Aconitase Activity

Aconitase activity and RNA-binding activity are mutually exclusive properties of IRP-1 (Basilion et al., 1994). Cytosolic aconitase activity rose by 40% with a 2-h iron pulse, but returned to base-line levels by 16 h (Table 2, 1a-1c). Cycloheximide failed to block the increase in aconitase activity (data not shown), consistent with an activation of IRP-1 (Tang et al., 1992). In contrast, chelation of iron by desferrioxamine lowered the cytosolic aconitase activity substantially (Table 2, 1d).



A 2-h incubation with ascorbate increased cytosolic aconitase activity by 2.5-fold (Table 2, 1 and 2a), again, not blocked by cycloheximide. Activity remained well above base-line levels even after a 16-h incubation (Table 2, 3a). When cells were incubated with ascorbate for 16 h and subsequently loaded with iron for 2 h, aconitase activity returned to the peak level (Table 2, 3b). The effect of ascorbate on cytosolic aconitase activity was less pronounced in cells preincubated with iron for 16 h (Table 2, 2a versus 2c). Dehydroascorbic acid, the oxidized form of the vitamin, also modulated cytosolic aconitase activity (Table 2, rows 4 and 5), indicating that the oxidation state of the vitamin in the medium was not crucial to the potentiation of aconitase activity.

Effect of Other Redox Compounds on Cytosolic Aconitase Activity

To investigate the role of the internal oxidative environment on cytosolic aconitase activity (Frei et al., 1988), we supplemented the medium with the powerful reducing agent uric acid (Sevanian et al., 1991). Incubation with uric acid for 2 or 16 h did not modify aconitase activity (Table 2, 6a and 7a). Preincubation with uric acid blocked the increase in aconitase activity produced by 2 h iron loading (Table 2, compare 1b and 7b). In contrast, aconitase activity increased substantially in cells loaded with iron and then incubated with uric acid (Table 2, compare 1c and 6c).

We further investigated the effect of antioxidant agents on cytosolic aconitase activity by incubating cells with oxidized (GSSG) or reduced (GSH) glutathione (Meister, 1994). Uptake of [^14C]GSH by the cells was linear. Both GSH and GSSG increased cytosolic aconitase activity at 2 h, although less than ascorbic acid (Table 2, 8a and 10a). Loading cells with iron for 2 h after a 16-h incubation with GSH or GSSG raised aconitase activity only modestly (Table 2, 9b and 11b), which also differed from the marked increase with ascorbate (Table 2, 3b). GSH and GSSG mimicked ascorbate when added to cells for 2 h after a 16-h preincubation with iron (Table 2, 2c, 8c, and 10c). The experiments with uric acid and glutathione indicate that, while alterations in the internal cellular oxidative environment can modify aconitase activity, ascorbic acid has additional actions.

Ascorbic Acid Increases Cytosolic Aconitase Activity without Affecting Ferritin mRNA Translation

Ferritin synthesis substantially exceeded control in cells incubated for 2 h in medium supplemented with 10 µg/ml ferric ammonium citrate (FAC), while aconitase activity rose by 1.5-fold (Fig. 1, lane3). Ferritin synthesis in cells incubated with FAC for 16 h exceeded that of the 2-h incubation (Fig. 1, lane4). Aconitase activity, however, returned to base-line levels, suggesting that blockade of translation by IRP-1 is separable from cytosolic aconitase activity.


Figure 1: The effect of iron, ascorbic acid, and desferrioxamine on de novo ferritin biosynthesis by K562 human erythroleukemia cells. A total of 2 10^7 cells were incubated in RPMI 1640 medium with various supplements. Half of the cells were used to measure cytosolic aconitase activity. Those remaining were metabolically labeled with [S]methionine, washed with PBS buffer three times, and solubilized in a buffer containing 1% Triton X-100, 0.15 M NaCl, 0.02 M Tris-HCl, pH 7.5, and 0.2 mM phenylmethylsulfonyl fluoride. The supernatant from a high speed spin was resolved on a 15% SDS -phosphate-urea gel. Each treatment was repeated several times. A representative result is shown. Lane1, ^14C-labeled marker proteins. Lane2, control cells. Cells incubated in medium supplemented with 10 µg/ml ferric ammonium citrate for 2 h (lane3) or 16 h (lane4). The cells in lane5 were treated with ferric ammonium citrate for 16 h, washed in PBS, and incubated for 2 h in medium supplemented with 150 µM ascorbate (AA). Cells incubated in medium supplemented with 150 µM ascorbate for 2 h (lane6) or 16 h (lane7). In lane8, the cells were treated with ascorbate for 16 h, washed, and incubated for 2 h in medium supplemented with 10 µg/ml ferric ammonium citrate. Lane9, cells incubated for 16 h in medium supplemented with 100 µM desferrioxamine (Des).



Two hours of 150 µM ascorbate did not change ferritin synthesis but increased cytosolic aconitase activity by 2.5-fold. Cytosolic aconitase activity declined at 16 h of ascorbate incubation, but still exceeded control by 1.5-fold with no alteration in ferritin synthesis. Preincubation of cells with ascorbic acid for 16 h followed by FAC for 2 h raised the level of ferritin translation (Fig. 1, lanes3 and 8). The obverse experiment, addition of ascorbate to cells preincubated with iron, increased both ferritin synthesis and aconitase activity (Fig. 1, lane5). Ascorbate demonstrated clearly the independent modification of cytosolic aconitase activity and ferritin synthesis.

Ferritin Synthesis, Cytosolic Aconitase Activity, and Antioxidants

Ferritin synthesis and aconitase activity were virtually unchanged in cells incubated with uric acid (Fig. 2, lanes6 and 7). Ferritin synthesis increased by about 2-fold and aconitase activity by about 1.5-fold in cells that were incubated sequentially with 10 µg/ml FAC for 16 h and with 500 µM uric acid for 2 h (Fig. 2, lanes4 and 5), changes that were less marked than with ascorbate (Fig. 1, lanes4 and 5). When the order of incubation was reversed, with uric acid followed by FAC, the increase in ferritin synthesis approximated that seen with ascorbic acid (Fig. 2, lanes7 and 8). In marked contrast to the ascorbate experiment in which aconitase activity rose by 2.5-fold, aconitase activity decreased in the cells treated with uric acid.


Figure 2: The effect of iron, uric acid, and desferrioxamine on de novo ferritin synthesis in K562 cells. Following the appropriate incubations, half the cells were metabolically labeled and the cytosolic supernatant was electrophoretically separated as in Fig. 1. Lane1, control. Lane2, 100 µM desferrioxamine for 16 h. Cells incubated with 10 µg/ml ferric ammonium citrate for 2 h (lane3) or 16 h (lane4). The cells in lane5 were incubated for 16 h in medium containing ferric ammonium citrate, after which they were washed and treated for an additional 2 h in medium supplemented with 500 µM uric acid (UA). Cells incubated in medium containing uric acid for 2 h (lane6) or 16 h (lane7). The cells in lane 8 were incubated for 16 h in medium containing uric acid, washed, and treated for another 2 h in medium containing ferric ammonium citrate. Half of each batch of cells was used to assay cytosolic aconitase activity.



Incubation with 500 µM GSSG changed neither ferritin synthesis nor cytosolic aconitase activity (Fig. 3, lanes7 and 8). Sequential treatment with iron and GSSG increased cytosolic aconitase activity by 50% without changing ferritin synthesis (Fig. 3, lanes4 and 5).


Figure 3: The effect of iron, oxidized (GSSG), and reduced (GSH) glutathione on de novo ferritin synthesis in K562 cells. The experimental protocol was similar to that detailed in Fig. 2except that uric acid was replaced with 500 µM reduced or oxidized glutathione in 100 mM HEPES buffer, pH 7.2.



GSH produced a different spectrum of changes, increasing ferritin synthesis but not aconitase activity (Fig. 3, lanes10 and 11). These data are the obverse of those obtained with ascorbate alone, which did not alter ferritin synthesis, but raised aconitase activity by severalfold. In aggregate, the data indicate that ferritin translation and cytosolic aconitase activity can be modified independently, suggesting a more complicated picture than one in which IRP-1 is either bound to message and enzymatically inactive or free in the cytosol with enzyme activity. The experiments with the antioxidants indicate that the effect of ascorbate involves more than its redox activity.

Mechanism by Which Ascorbate Increases Cytosolic Aconitase Activity

Ascorbate does not operate by dissociating IRP-1 from the ferritin message; although it increases aconitase activity, ferritin synthesis (which reflects the IRP-1/mRNA interaction) is unchanged. To determine whether ascorbate increased the quantity of IRP-1 with a secondary rise in aconitase activity, cytoplasmic extracts were prepared, separated electrophoretically, and used for immunoblotting with an antibody specific to IRP-1. As shown in Fig. 4, the cellular content of IRP-1 was uniform under each condition tested.


Figure 4: Detection of IRP-1 in K562 cells by Western blot analysis. After various treatments, cells were washed with PBS twice and lysed as described in Fig. 1. A 50-µg sample of mitochondrial-free cell lysate (100,000 g supernatant) from each treatment was separated by 10% SDS-PAGE, immobilized on a nitrocellulose membrane, and hybridized with rabbit antibody raised against the human IRP-1 (diluted 1:500). The antibody-antigen complex was visualized with goat antibody against rabbit IgG with coupled horseradish peroxidase (Bio-Rad) according to the supplier's manual. The antibody hybridized to a single protein in the 90-kDa range. Immunoblot analysis was performed three times, and one representative result is shown. Lane1, control. Lane2, 10^7 cells treated overnight with 100 µM desferrioxamine. Cells incubated in medium containing 10 µg/ml ferric ammonium citrate for 2 h (lane3) or 16 h (lane4). In lane5, cells were treated for 16 h in medium supplemented with iron, followed after washing by a 2 h treatment with 150 µM ascorbic acid. In another set of experiments cells were treated with 150 µM ascorbate for 2 h (lane6) or for 16 h (lane7). In lane8, cells incubated with ascorbate were loaded with iron for 2 h.



Ascorbate, therefore, activates enzymatically dormant aconitase protein. Ascorbate added in vitro to cytosolic extracts from control cells did not alter aconitase activity, indicating that the vitamin does not directly modify the aconitase protein nor act as an allosteric regulator. The ascorbate effect on cytosolic aconitase activity requires cell metabolism, but not protein synthesis.

Gel retardation experiments assessed the effect of ascorbic acid on the interaction of IRP-1 and the IRE. Cytosolic extracts from control cells and cells subjected to various treatments (see Fig. 5and legend) were incubated with a P-labeled IRE probe and then resolved electrophoretically, revealing the free IRE probe as well as the single band of the IREbulletIRP-1 complex.


Figure 5: The effect of desferrioxamine, iron, and ascorbic acid on the capacity of the IRP-1 to bind the IRE. IRE binding analysis was performed with control cells (lane1), cells incubated with 100 µM desferrioxamine 16 h (lane2), iron for 2 h (lane3) or 16 h (lane4), iron for 16 h followed by ascorbate for 2 h (lane5), ascorbate for 2 h (lane6) or 16 h (lane7), or ascorbate for 16 h followed by 2 h of iron (lane8). In each case, 2 µg of freshly isolated detergent extract was analyzed for IRE binding capacity with excess amounts of P-labeled probe (according to Leibold and Munro(1988) and Hirling et al.(1992)). The experiment was repeated several times, and one representative result is shown.



The extract from cells treated with desferrioxamine or iron behaved as expected, binding more or less of the IRE probe, respectively. Aconitase activity increased by 50% in cells loaded with iron for 16 h and subsequently incubated with ascorbate for 2 h (Fig. 1, lanes4 and 5). Electrophoretic mobility assay of IREbulletIRP-1 complex from comparably treated cells showed similar patterns in either circumstance, indicating that the increase in aconitase activity produced by ascorbate did not reflect a change in affinity between IRP-1 and the IRE (Fig. 5, lanes4 and 5).

Cells incubated with iron for 2 h after a 16-h exposure to ascorbate produced a more prominent signal by electrophoretic mobility shift assay than did cells merely incubated with ascorbate for 16 h, implying a greater affinity of IRP-1 for the IRE (Fig. 5B, lanes7 and 8). However, aconitase activity increased substantially under these conditions (Fig. 1, lanes7 and 8). Since the quantity of IRP in the cells was constant (Fig. 4, lane8), these data are most consistent with ascorbate activation of latent cytosolic aconitase.

Comparison of the electrophoretic mobility assay data and ferritin synthetic data further emphasizes the dissociation produced by ascorbate between IRE/IRP-1 affinity and translation of ferritin mRNA. The electrophoretic mobility patterns were virtually identical for cells treated with iron for 16 h and those where the treatment was followed by a 2-h incubation with ascorbate (Fig. 5, lanes4 and 5), while ferritin synthesis increased dramatically in the latter case (Fig. 1, lanes4 and 5). A similar dissociation between ferritin synthesis and IRE/IRP-1 affinity is seen with cells treated with ascorbate for 16 h and those where the treatment was followed by iron for 2 h (Fig. 5, lanes7 and 8versusFig. 1, lanes7 and 8). Fig. 5shows, if anything, a slight increase in the affinity between the IRE and IRP-1 in cells treated briefly with iron after ascorbate incubation, although this combination increased ferritin synthesis dramatically (Fig. 1).


DISCUSSION

Ascorbic acid is crucial to numerous cellular reactions, ranging from hydroxylation of lysyl -amino groups in collagen to the conversion of norepinephrine to epinephrine (Levine, 1988). Most cells in culture are ascorbate-deficient due to the lability of the vitamin in solution, disturbing a number of biochemical pathways, including lysosomal degradation of cytosolic ferritin (Englard and Seifter, 1986; Lipschitz et al., 1971). Our recent demonstration that ascorbate repletion greatly enhances ferritin synthesis in cells stimulated with iron indicated that a completely different arm of cellular metabolism is influenced by this vitamin (Toth et al., 1995).

We now show that ascorbate induces cytosolic aconitase activity in K562 cells, which are known to be ascorbate-deficient (Bridges and Hoffman, 1986). IRP-1 has been shown previously to acquire aconitase activity when released from the IRE in the 5`-untranslated region of the ferritin message (Haile et al., 1992). Ascorbate, in contrast, activates aconitase without changing ferritin synthesis, meaning that the vitamin does not dissociate IRP-1 from the ferritin message. The most reasonable conclusion is that the vitamin activates a cytoplasmic pool of enzymatically dormant aconitase protein.

Cells contain more than one IRP. The 90-kDa protein, designated IRP-1, is an aconitase when freed from the message (Constable et al., 1992; Müllner,et al., 1989; Goosen, et al., 1990). A second IRE-binding protein, of 110 kDa, termed IRP-2, lacks aconitase activity (Philpott et al., 1994; Henderson et al., 1993; Samaniego et al., 1994). One explanation of our data is that ascorbate induces aconitase activity in IRP-2. The data in Fig. 1argue against this possibility, however. If ascorbate triggered aconitase activity in IRP-2, then the 2.5-fold increase in activity with a 2-h incubation with the vitamin would reflect activation of IRP-2. The low rate of ferritin is consistent with IRP-1 remaining attached to the message. When cells preincubated with iron for 16 h are pulsed with ascorbate for 2 h, ferritin synthesis increases and aconitase activity rises by only 1.5-fold. If ascorbate imparted aconitase activity to IRP-2, enzymatic activity again should have increased by at least 2.5-fold over base-line levels. Furthermore, the increase in ferritin synthesis indicates that IRP-1 has detached from ferritin mRNA, which should have increased cytosolic aconitase activity even further.

We believe that the data are most consistent with ascorbate activation of latent IRP-1 in the cytosol (Fig. 6). Iron releases IRP-1 from the message. A second step, facilitated by ascorbate, converts this enzymatically inactive protein into an aconitase. Ascorbate stimulates aconitase activity in unbound IRP-1 but does not influence IRP-1 that is attached to the message. Eventually, the free IRP-1 reaches a new steady-state between active and inactive aconitase (Fig. 1, 16 h AA). When iron is added to these cells for 2 h, it releases IRP-1 into a smaller pool of free, inactive IRP-1. A larger quantity of IRP-1 is released before steady state is reached, so that both aconitase activity and ferritin synthesis increase (Fig. 1, 16 h AA + 2 h Fe). The three-state model presented in Fig. 6would allow independent regulation of the two activities of IRP-1, permitting greater flexibility in the regulation of the metabolic environment of the cell than does a model in which the IRP-1 either is bound to mRNA with no aconitase activity or free in the cytosol with aconitase activity.


Figure 6: Schematic representation of the role of ascorbate in ferritin synthesis. IRP-1 is released from the message by iron (step1). The cytosolic IRP-1 initially lacks aconitase activity. The enzymatic activation of IRP-1 is facilitated by ascorbate (step2). Superoxide inactivates the aconitase capacity of IRP-1.



Further biochemical studies are required to determine the molecular steps by which ascorbate switches IRP-1 into the aconitase mode. The recent demonstration that cytosolic aconitase is inactivated by superoxide (Gardner and Fridovich, 1992; Gardner and Fridovich, 1993) suggests that ascorbate could modulate the activity of this enzyme, at least in part, by buffering cytoplasmic superoxide. Nitric oxide has been posited to be a physiological regulator of cytosolic aconitase activity (Weiss et al., 1993). The recent data indicating that superoxide and other free radicals, rather than nitric oxide, mediate cytosolic aconitase activity highlight the importance of identifying the physiologically relevant biochemical constituents in the cell (Hausladen and Fridovich, 1994). The cellular physiology of iron and the plethora of proteins that depend on it cannot be understood without this information.


FOOTNOTES

*
This work was supported by National Institutes of Health Grant HL 45794. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
To whom correspondence should be addressed: Division of Hematology-Oncology, Brigham and Women's Hospital, 75 Francis St., Boston, MA 02115. Tel.: 617-732-5842; Fax: 617-739-3324; bridges{at}calvin.bwh.harvard.edu.

(^1)
The abbreviations used are: IRE, iron-responsive element; PBS, phosphate-buffered saline; FAC, ferric ammonium citrate.


ACKNOWLEDGEMENTS

We are grateful to Drs. H. Franklin Bunn, Jack T. Rogers, and Gabor Lazar for their valuable input over the course of this study, as well as to Dr. L. C. Kühn for the generous gifts of antibody to IRP-1 and IRE probe. Suzanne M. Elliott and Jay A. McPhee provided expert technical assistance during the course of these experiments. Part of the work was done by Kulleni Gebreyes during a summer internship.


REFERENCES

  1. Basilion, J. P., Rouault, T. A., Massinople, C. M., Klausner, R. D., and Burgess, W. H. (1994) Proc. Natl. Acad. Sci. U. S. A. 91,574-578 [Abstract]
  2. Bridges, K. R. (1987) J. Biol. Chem. 262,14773-14778 [Abstract/Free Full Text]
  3. Bridges, K. R., and Hoffman, K. E. (1986) J. Biol. Chem. 261,14273-14277 [Abstract/Free Full Text]
  4. Burns, J. J. (1957) Nature 180,553-555
  5. Constable, A., Quick, S., Gray, N. K., and Hentze, M. W. (1992) Proc. Natl. Acad. Sci. U. S. A. 89,4554-4558 [Abstract]
  6. Emery-Goodman, A., Hirling, H., Scarpellino, L., Henderson, B., and Kühn, L. (1993) Nucleic Acids Res. 21,1457-1461 [Abstract]
  7. Englard, S., and Seifter, S. (1986) Annu. Rev. Nutr. 6,365-406 [CrossRef][Medline] [Order article via Infotrieve]
  8. Frei, B., Stocker, R., and Ames, B. N. (1988) Proc. Natl. Acad. Sci. U. S. A. 85,9748-9752 [Abstract]
  9. Gardner, P. R., and Fridovich, I. (1992) J. Biol. Chem. 267,8757-63 [Abstract/Free Full Text]
  10. Gardner, P. R., and Fridovich, I. (1993) Arch. Biochem. Biophys. 301,98-102 [CrossRef][Medline] [Order article via Infotrieve]
  11. Goosen, B., Caughman, S. W., Harford, J. B., Klausner, R. D., and Hentze, M. W. (1990) EMBO J. 9,4127-4133 [Abstract]
  12. Haile, D. J., Rouault, T. A., Harford, J. B., Kennedy, M. C., Blondin, G. A., Beinert, H., and Klausner, R. D. (1992) Proc. Natl. Acad. Sci. U. S. A. 89,11735-11739 [Abstract]
  13. Harrell, C. M., McKenzie, A. R., Patio, M. M., Walden, W. E., and Theil, E. C. (1991) Proc. Natl. Acad. Sci. U. S. A. 88,4166-4170 [Abstract]
  14. Hausladen, A., and Fridovich, I. (1994) J. Biol. Chem. 269,29405-29408 [Abstract/Free Full Text]
  15. Henderson, B. R., Seiser, C., and Kühn, L. C. (1993) J. Biol. Chem. 268,27327-27334 [Abstract/Free Full Text]
  16. Hentze, M. W., Caughman, S. W., Casey, J. L., Koeller, D. M., Rouault, T. A., Harford, J. B., and Klausner, R. D. (1988) Gene (Amst.) 72,201-208 [CrossRef][Medline] [Order article via Infotrieve]
  17. Hirling, H., Henderson, B. R., and Kühn, L. C. (1994) EMBO J. 13,453-461 [Abstract]
  18. Hoffman, K. E., Yanelli, K., and Bridges, K. R. (1991) Am. J. Clin. Nutr. 199,1188S-1192S
  19. Kühn, L. C., and Hentze, M. W. (1992) J. Inorg. Biochem. 47,183-195 [CrossRef][Medline] [Order article via Infotrieve]
  20. Levine, M. (1988) N. Engl. J. Med. 314,892-902 [Medline] [Order article via Infotrieve]
  21. Liebold, E. A., and Munro, H. N. (1988) Proc. Natl. Acad. Sci. U. S. A. 85,2171-2175 [Abstract]
  22. Lipschitz, D. A, Bothwell, T. H., Seftel, H. C., Wapnick, A. A., Charlton R. W. (1971) Br. J. Haematol. 20,155-63 [Medline] [Order article via Infotrieve]
  23. Meister, A. (1994) J. Biol. Chem. 269,9397-9400 [Free Full Text]
  24. Moser, U., and Bendich, A. (1991) in Handbook of Vitamins (Machlin, L. J., ed) pp. 195-232, Marcel Dekker, Inc., New York
  25. Müllner, E. W., Neupert, B., and Kühn, L. C. (1989) Cell 58,373-382 [Medline] [Order article via Infotrieve]
  26. Nishikimi, M., and Yagi, K. (1991) Am. J. Clin. Nutr. 54,1203S-1208S [Abstract]
  27. Philpott, C. C., Haile, D., Rouault, T. A., and Klausner, R. D. (1993) J. Biol. Chem. 268,17655-17658 [Abstract/Free Full Text]
  28. Philpott, C. C., Klausner, R. D., and Rouault, T. A. (1994) Proc. Natl. Acad. Sci. U. S. A. 91,7321-7325 [Abstract]
  29. Rouault, T. A., Hentze, M. W., Caughman, S. W., Harford, J. B., and Klausner, R. D. (1988) Science 241,1207-1210 [Medline] [Order article via Infotrieve]
  30. Samaniego, F., Chin, J., Iwai, K., Rouault, T. A., and Klausner, R. D. (1994) J. Biol. Chem. 269,30904-30910 [Abstract/Free Full Text]
  31. Sevanian, A., Davies, K. J. A., and Hochstein, P. (1991) Am. J. Clin. Nutr. 54,1129S-1134S [Abstract]
  32. Smith, L. (1955) Methods Biochem. Anal. 2,427-433 [Medline] [Order article via Infotrieve]
  33. Szent-Györgyi, A. (1928) Biochem. J. 22,1387-1409
  34. Tang, C. K., Chin, J., Harford, J. B., Klausner, R. D., and Rouault, T. A. (1992) J. Biol. Chem. 267,24466-24470 [Abstract/Free Full Text]
  35. Toth, I., Rogers, J. T., McPhee, J. A., Elliott, S. M., Abramson, S. L., and Bridges, K. R. (1995) J. Biol. Chem. 270,2846-2852 [Abstract/Free Full Text]
  36. Weiss, G., Goossen, B., Doppler, W., Fuchs, D., Pantopoulos, K., Werner-Felmayer, G., Wachter, H., and Hentze, M. W. (1993) EMBO J. 12,3651-3657 [Abstract]

©1995 by The American Society for Biochemistry and Molecular Biology, Inc.