Iron Loading into Ferritin by an Intracellular Ferroxidase

ARCHIVES OF BIOCHEMISTRY AND BIOPHYSICS

Vol. 359, No. 1, November 1, pp. 69 –76, 1998 Article No. BB980891

Iron Loading into Ferritin by an Intracellular Ferroxidase Christopher A. Reilly and Steven D. Aust1 Biotechnology Center, Utah State University, Logan, Utah 84322-4705

Received June 1, 1998, and in revised form August 4, 1998

An intracellular, membrane-bound enzyme exhibiting both p-phenylenediamine oxidase activity and ferrous iron oxidase activity was isolated with the plasma membrane fraction of horse heart and studied for its ability to load iron into ferritin. The ferroxidase activity of the tissue oxidase was stimulated approximately twofold by horse spleen apoferritin, and the iron was loaded into ferritin. The loading of iron into ferritin by the tissue oxidase was inhibited by antihorse serum ceruloplasmin antibody. The stoichiometry of iron oxidation and oxygen consumption during iron loading into ferritin by the tissue-derived oxidase and serum ceruloplasmin were 3.6 6 0.2 and 3.9 6 0.2, respectively. These data provide evidence that an enzyme analogous to ceruloplasmin is present on the plasma membrane of horse heart and that this ferroxidase is capable of catalyzing the loading of iron into ferritin. The implications of these data on the present models for the uptake and storage of iron by cells are discussed. © 1998 Academic Press

Recently, a significant amount of research has been performed to better understand cellular iron metabolism. Several findings suggest that copper plays an integral role in the cellular homeostasis of iron. Defects in copper metabolism as well as dietary copper deficiency have been shown to have profound effects on cellular and systemic iron homeostasis (1–3). For example, dietary copper deficiency has been shown to induce anemia that was not ameliorated by iron supplementation; however the condition was completely reversed upon copper supplementation (2, 3). In addition, a direct involvement of the copper-containing enzyme ceruloplasmin in iron metabolism is evident from studies on individuals with aceruloplasminemia, a genetic disorder that results in a deficiency in active 1 To whom correspondence should be addressed. Fax: (435) 7972755. E-mail: [email protected].

0003-9861/98 $25.00 Copyright © 1998 by Academic Press All rights of reproduction in any form reserved.

ceruloplasmin (1, 4 – 8). Patients with aceruloplasminemia exhibit characteristic opthalmic and neurological degeneration as well as systemic iron deposition and tissue damage (1, 4 – 8). Under normal conditions, iron is stored as a soluble and stable complex within ferritin (9, 10). Ferritin is a ubiquitous 450- to 500-kDa intracellular iron storage protein where up to 2500 atoms of iron can be stored as a ferric oxyhydroxy phosphate complex (11). Many researchers have proposed that the mechanism of iron loading into ferritin is related to a ferroxidase center located on the H subunits of ferritin (9, 10, 12, 13). We do not believe that this is the physiological mechanism for loading iron into ferritin. We found that the ferroxidase activity of ferritin occurred only in the presence of a Good buffer (14). In addition, the stoichiometry of iron loading using this system was two moles of iron oxidized per mole dioxygen reduced, indicating that hydrogen peroxide was produced (15, 16). In the presence of ferrous iron, H2O2 is reduced to produce the hydroxyl radical, a nonspecific oxidant of biological macromolecules. Ferritin loaded using its ferroxidase activity exhibited properties which differed significantly from preparations of native ferritin (14). Iron has also been loaded into ferritin enzymatically using ceruloplasmin (14, 17). Ceruloplasmin catalyzes the oxidation of ferrous iron with the concomitant incorporation of iron into ferritin and the complete fourelectron reduction of dioxygen to water (16). Ferritin loaded using ceruloplasmin was found to have properties identical to those observed for native ferritin (14). Iron loading into ferritin by ceruloplasmin has also been shown to be a site-specific process, occurring only at the H subunits of ferritin (18). It has been shown that only the H subunit of ferritin can stimulate the ferroxidase activity of ceruloplasmin and that the strength of the interaction between ferritin and ceruloplamsin is specific for the species from which the proteins were isolated (18, 19). In addition, the loading of iron into ferritin required the presence of an open a-helix bundle channel which traversed the outer shell 69

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of ferritin and allowed iron to enter the core; genetic engineering of an opened a-helix bundle channel in L homopolymers of ferritin permitted iron loading; however, the ferroxidase activity of ceruloplasmin was not affected (20). Historically, ceruloplasmin has been considered to be strictly a serum protein, synthesized in the liver and secreted into the blood (21). However, iron loading into ferritin must occur within cells. There have been numerous reports on the expression and induction of ceruloplasmin mRNA in nonsecretory tissues, including the retina (22), placenta, testis (23), heart (24), lungs (25),and brain (26, 27). In most instances, the induction of mRNA was observed in response to physical injury and/or exposure to redox-active chemicals. These data are consistant with ceruloplasmin being an acute-phase protein. Recently, however, it has been reported that ceruloplasmin was present on the plasma membrane of astrocytes and was proposed to be instrumental in iron homeostasis in the brain (28). Collectively, these studies suggest that ceruloplasmin or a ceruloplasmin-like protein exists in cells. The purpose of this investigation is to isolate a ferroxidase from tissue and to assess the ability of this enzyme to load iron into ferritin. MATERIALS AND METHODS Chemicals. Ferrozine [3-(2-pyridyl)-5,6-bis(4-phenylsulfonic acid)-1,2,4-triazine], Chelex 100 (Bio-Rad Laboratories, Richmond, CA), sodium acetate, Tris base, trichloroacetic acid, ferrous chloride, thioglycolic acid, e-amino-n-caproic acid, a,a9-dipyridyl and 2,6dichlorophenol-indophenol, N,N,N9,N9-tetramethyl-p-phenylenediamine, ouabain, adenosine 59-triphosphate, bathocuproinedisulfonic acid, and bathophenanthroline were purchased from Sigma Chemical Co. (St. Louis, MO). Ferrous ammonium sulfate, manganese chloride, calcium chloride, magnesium chloride, aluminum chloride, sodium succinate, potassium phosphate, and sodium oxalate were purchased from Mallinkrodt (Paris, KY); L-histidine and p-phenylenediamine from Eastman Kodak (Rochester, NY), and sodium azide, disodium ethylenediamine tetracetic acid, and potassium ferricyanide from Fisher Scientific Co. (Fairlawn, NJ). Anti-horse serum ceruloplasmin antiserum was prepared as a service provided by the Macromolecular Services Laboratory at Utah State University. All solutions used for oxidase assays were prepared in purified water (Barnstead NANOpure Infinity System; specific resistance .17 mV/ cm) and further treated to remove contaminating transition metals by chromatography over Chelex 100 chelating resin. Protein purification and preparation. Ceruloplasmin (EC 1.16.3.1) was isolated from horse serum purchased from Pel-Freez Biologicals (Rogers, AR) as previously described (29). Ferritin was isolated from horse spleen purchased from Pel-Freez Biologicals, as described by deSilva et al. (11). Apoferritin was prepared by reduction of the iron in ferritin using 5% w/v thioglycolic acid in 50 mM sodium acetate, pH 5.5, and chelating the ferrous iron with a,a9dipyridyl, as previously described (30). The amount of iron present in ferritin was determined using the total iron assay described by Brumby and Massey (31). The tissue-derived oxidase was isolated from horse heart purchased from Pel-Freez Biologicals. Typically, 50 –100 g of horse heart was washed with 0.9% saline, minced with scissors, and homogenized in 100 mM NaCl, pH 7.0, containing 20 mM e-amino-n-caproic

acid using a Potter–Elvehjem homogenizer. The tissue homogenate was centrifuged at 500g and 4°C for 10 min. The supernatant was decanted and membrane pelleted by centrifugation at 20,000g and 4°C for 30 min. The resulting pellet was resuspended in 100 mM NaCl, pH 7.0, containing 20 mM e-amino-n-caproic acid and sonicated for five 15-s cycles using a Branson Sonifier 250. The preparation was again centrifuged at 20,000g for 15 min and the supernatant concentrated using an Amicon stirred-cell concentrator fitted with a PM-30 ultrafiltration membrane (Amicon Inc., Beverly, MA). The sample was then chromatographed over a Sepharose CL-6B gel filtration column and eluted with 100 mM NaCl, pH 7.0, containing 20 mM e-amino-n-caproic acid. Fractions eluting from the column were assayed for p-phenylenediamine oxidase activity, as previously described (32), and pooled. Briefly, samples were incubated with 10 mM p-phenylenediamine in 100 mM sodium acetate, pH 5.0, and 37°C. Oxidation of p-phenylenediamine was determined spectrophotometrically as an increase in absorbance at 570 nm. Absorbances were determined using an EL311 microplate reader (BioTek Instruments, Winoski, VT) using dual-wavelength detection at 570 and 630 nm. One unit of activity was defined as the amount of oxidase required to cause an absorbance change of 1.0 at 570 nm using the conditions described above. The concentration of protein was determined by the Bradford assay using bovine serum albumin (BSA) as a standard. Assays for marker enzymes. The origin of the membrane fraction was determined by the presence of marker enzyme activities. Plasma membrane was identified by the presence of ouabain-sensitive Na1/K1 ATPase activity (EC 3.6.1.3) and assayed as described by Bonting et al. (33) and the liberated phosphate was quantitated using the method of Taussky and Shorr (34). The mitochondrial enzyme succinate dehydrogenase (EC 1.3.99.1) was assayed as described by Earl and Korner (35), except that 2,6-dichlorophenol indophenol was used. The presence of sarcoplasmic reticulum was determined using the Ca21 loading assay described by Meissner (36), with modifications. Calcium loading was monitored by the decrease in calcium remaining in solution and as the increase in calcium associated with the vessicles as detected by inductively coupled plasma-mass spectrometry. Ferroxidase activity and iron loading into ferritin. The ability of ceruloplasmin and the tissue-derived oxidase to oxidize ferrous iron was determined using the discontinuous ferrozine assay, as described previously (19). Briefly, samples were incubated at 37°C in 50 mM NaCl, pH 7.0, and in the presence or absence of horse spleen apoferritin. Reactions were initiated by the addition of histidinechelated ferrous iron [His:Fe(II) (5:1)], prepared in argon-purged 50 mM sodium chloride, pH 7.0. At specific time points, 100-mL aliquots were removed and added to 900 mL of 2.5 mM ferrozine. The amount of ferrous iron remaining in solution was determined by spectrophotometric detection of the ferrous:ferrozine complex (e570 5 27,900 M21 cm21) and the extent of iron oxidation determined as the difference in absorbance for identical incubations with and without enzyme. Measurement of oxygen consumption during iron loading into ferritin was performed using a Gilson Oxy5/6 oxygraph equipped with a Clarke-type electrode. Iron loading into apoferritin was performed as described by deSilva et al. (14). Briefly, equimolar concentrations of apoferritin and ceruloplasmin or an equivalent amount of the tissue-derived oxidase activity were incubated at 37°C in 50 mM NaCl, pH 7.0. Loading was initiated by the addition of histidine-chelated ferrous iron [His:Fe(II) 5:1] and monitored spectrophotometrically as an increase in absorbance at 380 nm. Loading reactions were terminated by the addition of 1 mM ferrozine and chromatographed over an Econo-Pac 10 DG column (Bio-Rad Laboratories) to remove nonprotein bound iron. The quantity of iron in ferritin was determined using the total iron assay (31). The presence of iron in ferritin was also confirmed by native PAGE using a 7.5% Tris–HCl Ready Gel (Bio-Rad Laboratories) and

71

IRON LOADING INTO FERRITIN BY FERROXIDASE TABLE I

Purification of a Tissue-Derived Oxidase

Homogenate 20,000g supernatant 20,000g pellet Sepharose CL-6B

Volume (mL)

Protein (mg/mL)a

Specific activitya

Total activitya

Fold purification

350 275 30 8.5

7.2 7.2 5.5 1.8

1.0 N.D. 6.2 23.0

2270 N.D. 1000 350

– – 6.2 23.0

a

Oxidase activity was determined using the p-phenylenediamine oxidase assay as described under Materials and Methods. Protein concentration was determined by the Bradford assay.

staining for iron with 0.1% w/v potassium ferricyanide in 1 N hydrochloric acid (18).

RESULTS

inhibited by aluminum and azide. Aluminum has previously been shown to compete with iron for the binding of the active site of ceruloplasmin (37), while azide effects the type-I copper liganding in ceruloplasmin

Purification of tissue ferroxidase. Horse heart homogenate exhibited substantial p-phenylenediamine oxidase activity (Table I). Activity due to contamination with serum ceruloplasmin was deemed negligable since the presence of approximately 4 mL of serum would be required to generate the activity present in 1 cm3 of tissue. The oxidase activity was pelleted upon centrifugation at 20,000g for 30 min. The pelleted membrane exhibited substantial ouabain-sensitive Na1/K1ATPase activity, approximately 3.1 mol of inorganic phosphate was liberated per milligram of protein in 1 h, but succinate dehydrogenase and Ca21 sequestration activities were not observed (data not shown). Based on the marker enzyme assays, it was concluded that the oxidase was located in the plasma membrane. The purification procedure resulted in a 23-fold purification; however, homogeneity was not obtained. Methods typically used to isolate membranebound proteins (i.e., n-butanol extraction, detergent solubilization, phospholipase A digestion, and limited proteolysis) resulted in the immediate inactivation of the enzyme. It was found that the rate of substrate oxidation was proportional to the concentration of membrane protein (Figs. 1A and 1B). When ferrous iron was used as the substrate, the membrane-bound oxidase exhibited typical Michaelis–Menton kinetics with a Km of 9 6 1.8 mM (Fig. 2). The Km for p-phenylenediamine and N,N,N9,N9-tetramethyl-p-phenylenediamine were 7 6 1.5 mM and 0.5 6 0.1 mM, respectively (data not shown). The pH optimum for the oxidation of p-phenlendiamine was 5.9 6 0.1 for both the tissue-derived ferroxidase and serum ceruloplasmin (data not shown). Inhibition of tissue ferroxidase. The p-phenylenediamine oxidase activity of both the tissue-derived oxidase and serum ceruloplasmin were similarly affected by various inhibitors (Table II). Both the tissue-derived oxidase and serum ceruloplasmin were significantly

FIG. 1. The effect of membrane protein concentration on the rate of substrate oxidation. (A) the rate of p-phenylenediamine oxidation as a function of membrane protein concentration. Experimental conditions were as described under Materials and Methods; however, the absorbance was determined after a 20-min incubation at 37°C. (B) the rate of ferrous iron oxidation as a function of membrane protein concentration. The experimental conditions were 40 mM ferrous ammonium sulfate, 50 mM NaCl, pH 7.0, and increasing concentrations of membrane protein. Ferrous iron oxidation was quantitated as described under Materials and Methods.

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FIG. 2. Kinetics of iron oxidation by the tissue-derived ferroxidase as a function of ferrous iron concentration. The experimental conditions were 0.3 U tissue-derived oxidase ( p-phenylenediamine oxidase activity), 50 mM NaCl, pH 7.0, and increasing concentrations of ferrous ammonium sulfate. Reactions were initiated by addition of iron, incubated for 1 min at room temperature, and terminated by the addition of 2.5 mM ferrozine. Iron oxidation was quantitated as described under Materials and Methods.

(38). In most cases, the extent of inhibition of the two activities were proportional. Iron loading into ferritin. In the presence of horse spleen apoferritin, the ferroxidase activity of both the tissue-derived oxidase as well as serum ceruloplasmin was stimulated approximately twofold (Fig. 3). Stimu-

TABLE II

Inhibition of the p-Phenylenediamine Oxidase Activity of a Tissue-Derived Oxidase and Serum Ceruloplasmin Percentage remaining activitya

Condition No additions 1 mM Bathocuproinedisulfonic acid 1 mM EDTA 10 mM Azide 50 mM AlCl3 100°C for 5 min

Tissue-derived oxidaseb

Serum ceruloplasminb

100

100

65 6 18 82 6 6 5.4 6 0.9 70 6 3 N.D.

52 6 17 65 6 7 N.D. 67 6 2 N.D.

a Activity was determined as the increase in absorbance at 570 nm due to the oxidation of p-phenylenediamine, as described under Materials and Methods. Data are means of triplicate assays with standard deviations. b Approximately 0.2 U of tissue ferroxidase or serum ceruloplasmin was used.

FIG. 3. The effects of horse spleen apoferritin on the oxidation of ferrous iron by the tissue-derived ferroxidase and serum ceruloplasmin. Reaction mixtures contained the necessary concentration of membrane protein to yield an equivalent amount of p-phenylenediamine oxidase activity as observed for 0.1 mM serum ceruloplasmin in the presence (F) or absence (Œ) of 0.1 mM horse spleen apoferritin, or 0.1 mM serum ceruloplasmin in the presence (■) or absence () of apoferritin, and 50 mM NaCl, pH 7.0. Reactions were initiated by addition of histidine-chelated ferrous iron (5:1) and maintained at 37°C. Iron oxidation was quantitated as described under Materials and Methods.

lation of the ferroxidase activity of serum ceruloplamsin by apoferritin has been previously described (18, 19). Addition of ferrous iron to samples containing the tissue-derived ferroxidase or serum ceruloplasmin and horse spleen apoferritin resulted in an increase in absorbance at 380 nm, indicative of the formation of loaded ferritin (Fig. 4). The loading of iron into ferritin was inhibited by several of the conditions listed in Table II (data not shown). Sequential addition of ferrous iron increased the extent of iron loaded into ferritin, indicating that the activity was not destroyed upon iron oxidation (Fig. 4). The extent of iron loading into ferritin by either the tissue-derived ferroxidase or serum ceruloplasmin was similar, but the rate of iron loading by serum ceruloplasmin was significantly faster. The total iron assay of ferritin loaded using the tissue-derived ferroxidase showed that approximately 75 6 6% of the total iron added was present in the high-molecular-weight fraction. The presence of iron in ferritin was confirmed by native PAGE and staining for iron (Figs. 5A and 5B). Similar results were obtained with ferritin loaded with iron using either the tissuederived ferroxidase or serum ceruloplasmin. The stoichiometry of iron oxidation to dioxygen reduction during iron loading into ferritin was 3.6 6 0.2 and 3.9 6 0.2 for the tissue-derived ferroxidase and serum ceru-

73

IRON LOADING INTO FERRITIN BY FERROXIDASE TABLE III

Stoichiometry of Iron Oxidation during Iron Loading into Apoferritina

Enzyme Tissue-derived ferroxidase Serum ceruloplasmin

Oxygen consumed

Iron oxidized

Ratio (Fe(III)/O2)

20 6 1

73 6 1

3.6 6 0.2

42 6 6

160 6 20

3.9 6 0.2

a Reaction conditions were identical to those described under Fig. 3, except that the concentration of serum ceruloplasmin and apoferritin were 1.0 mM. The extent of iron oxidation and oxygen consumption was determined as described under Materials and Methods and the ratio calculated at the 2.5-min time point. Data are means of triplicate assays with standard deviations.

FIG. 4. Sequential loading of iron into horse spleen apoferritin by the tissue-derived ferroxidase and serum ceruloplasmin. The experimental conditions were identical to those described in the legend for Fig. 3, except reactions were monitored spectrophotometrically for the formation of loaded ferritin as an increase in absorbance at 380 nm. The arrows represent the addition of ferrous iron.

serum resulted in the near complete loss of the ability of either enzyme to catalyze iron loading into ferritin (Figs. 6A and 6B). The decrease in the rate of iron loading by serum ceruloplasmin when compared to Fig. 4 was a result of storage and some proteolytic degradation of the enzyme. DISCUSSION

loplasmin, respectively (Table III). The migration of ferritin in native PAGE was not affected by iron loading, thus the charge/mass ratio of ferritin was not altered. These data indicate that no deleterious, partially reduced species of oxygen were produced during loading. Incubation of the tissue-derived ferroxidase and ceruloplasmin with anti-horse ceruloplasmin anti-

FIG. 5. Native PAGE of ferritin loaded with iron using the tissuederived ferroxidase or serum ceruloplasmin. (A) Native PAGE of ferritin loaded with iron and stained for the presence of iron using ferricyanide. Lane 1 is a standard of purified horse spleen ferritin and lane 2 is a standard of horse spleen apoferritin. In lanes 3 and 4, apoferritin was loaded with iron using the tissue-derived ferroxidase or serum ceruloplasmin, respectively. Lane 5 contains a standard of histidine-chelated ferric iron. (B) Ferritin subjected to native PAGE and stained for protein. Lane 1 is a standard of apoferritin. Lanes 2 and 3 contain apoferritin loaded with iron by the tissue-derived ferroxidase or serum ceruloplasmin, respectively.

The results obtained in this study demonstrate the presence of a cellular ferroxidase for the loading of iron into ferritin. Although purification of the tissue-derived ferroxidase to homogeneity was not achieved, characterization of the oxidase activity of the tissue enzyme indicated that it was analogous to serum ceruloplasmin. These findings demonstrate the presence of ceruloplasmin or a ceruloplasmin like protein associated with the plasma membrane. It has previously been suggested that ferritin and ceruloplasmin associate to form a protein–protein complex for iron loading into ferritin (39, 40). The presence of various synthetic peptides derived from the sequences of either ceruloplasmin or ferritin inhibited iron loading into ferritin by interfering with complex formation (40). Therefore, any substance that interferes with complex formation will inhibit iron loading into ferritin by ceruloplasmin. In our experiments using anti-ceruloplasmin antibody, the loading of iron into ferritin by either the tissue-derived ferroxidase or serum ceruloplasmin was almost completely inhibited. In addition, the rate of iron loading by the particulate enzyme was kinetically slower and only 75 6 6% of the iron was loaded into ferritin, approximately 20% lower than when serum ceruloplasmin was used. These data suggest that the tissue-derived ferroxidase also requires the formation of a protein–protein complex to load iron into ferritin; however, its particulate nature seems to affect this process. An additional factor that may influence the rate and efficiency of iron loading by

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FIG. 6. Inhibition of iron loading into apoferritin by anti-horse ceruloplasmin antibody. The experimental conditions were identical to those described in the legend for Fig. 4, except that ceruloplasmin (A) and the tissue-derived ferroxidase (B) were incubated for 24 h at 4°C in 0.5 M NaCl, pH 7.0, with 20 mL of anti-horse ceruloplasmin anti-serum. Horse spleen apoferritin (1 mM) was added to the samples prior to the addition of histidine-chelated ferrous iron. Iron loading due to endogenous ferroxidase activity of the anti-serum was subtracted from the data.

the particulate ferroxidase is the chelator of the ferrous iron. We used histidine-chelated ferrous iron at a 5:1 molar ratio. Unfortunately, the true physiological chelator(s) of ferrous iron is(are) not known. In our studies we found that elevated concentrations of histidine inhibited iron loading into ferritin by the particulate enzyme (data not shown). Therefore, the decreased rate and efficiency in iron loading exhibited by the particulate enzyme can be attributed to two factors: the spatial hindrance in complex formation with ferritin in vitro and the use of histidine as the ferrous iron chelator. Incubation of the membrane vesicles with proteases (i.e., proteinase k, subtilisin, and a-chymotrypsin), bee venom, and heating all readily inactivated the tissuederived oxidase. In addition, competitive and uncompetitive inhibitors of ceruloplasmin inhibited the tissue-derived ferroxidase to the same extent as with serum ceruloplasmin (Table II). These data suggest

that a similar active constituent and/or protein must be present. The membrane-bound oxidase, like ceruloplasmin, also exhibited a broad substrate specificity, with an apparent preference for ferrous iron (41). In addition, the stoichiometry of iron oxidation by both the tissue-derived ferroxidase and ceruloplasmin was approximately 4:1. Broad substrate specificity and a 4:1 stoichiometry for iron oxidation to oxygen reduction are both features common among multi-copper oxidases (32). Collectively, these data suggest that the tissue-derived oxidase is probably a member of the multi-copper oxidase family. Treatment of cells in culture with aluminum has been shown to induce microcytic anemia that was not reversible by iron supplementation (42). In addition, cellular iron accumulation without the storage of iron in ferritin was also observed (42). Similar observations have been made on individuals with aceruloplasminemia, Wilson’s disease, and in animals subject to dietary copper deficiency (1– 8). The inhibition of p-phenylenediamine oxidation and iron loading into ferritin by the tissue-derived oxidase and ceruloplasmin by aluminum suggests that the disruption of iron homeostasis due to aluminum overload may be a result of the inhibition of a copper-containing ferroxidase that is essential for normal cellular iron metabolism. In Saccharomyces cerevisia, a membrane-bound multi-copper oxidase (Fet3) is a necessary constituent of the high-affinity iron uptake system (reviewed in 43– 45). In this system, Fet3 acts in concert with FTR1 to oxidize and transfer iron across the cell membrane. The catalytic properties of Fet3 were found to be similar to mammalian ceruloplasmin (32), as well as the tissue ferroxidase described in this study. A second component of the yeast high-affinity iron-uptake system is a membrane-bound ferrireductase (Fre1 or Fre2) which reduces ferric iron and supplies ferrous iron to Fet3 prior to translocation of the iron into cells (43– 45). Recent studies indicate that mammalian cells treated with citrate- or NTA-chelated ferric iron require the reduction of iron prior to absorption (46, 47). In fact, NADH-dependent ferrireductase activity has been observed with plasma membrane prepared from K562 cells (48), HeLa cells (47, 49), skin fibroblasts (47), and hepatocytes (49), and partially purified from duodenal mucosal cells (50). Ferrireductase activity was also present on the membrane used in this study. Therefore, it may be possible that the ferroxidase presented in this study functions in a similar manner as Fet3 in the uptake of iron by mammalian cells. Studies have shown that nontransferrin-bound iron absorbed by cells ultimately accumulates in ferritin (51). It has also been shown that when iron is loaded into ferritin in the presence of intracellular concentrations of phosphate that the resulting iron core is significantly different from native ferritin. This has

IRON LOADING INTO FERRITIN BY FERROXIDASE

prompt some researchers to propose that ferritin must obtain its iron at a membrane-bound site within cells where the concentration of phosphate is low (52). As such, we propose that the ferroxidase described in this study may be functional in the absorption and storage of iron by cells and that the plasma membrane or endocytic vesicles derived from the plasma membrane may be the site of iron loading into ferritin. Coupling the enzymatic reduction of iron with the translocation and loading of iron into ferritin by a membrane-bound ferroxidase is a logical mechanism for the safe handling iron, preventing deleterious oxidations catalyzed by iron and the precipitation of ferric iron. In summary, a ferroxidase analogous to serum ceruloplasmin and yeast Fet3 has been shown to be associated with the plasma membrane of cardiac muscle. These findings may help broaden our understanding of how iron is loaded into ferritin in vivo, as well as assist in the formulation of more suitable models used to describe the mechanism of cellular iron absorption. Although the tissue ferroxidase could be not identified, efforts are currently underway to purify this protein to homogeneity. ACKNOWLEDGMENTS The authors thank Terri Maughan for her secretarial assistance in preparation of the manuscript and James Grace for technical assistance and helpful discussions. This work was supported by NIH Grants ES05056 and DK52823.

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