Detection and Isolation of a Hepatic Membrane Receptor for Ferritin*

A ferritin receptor has been detected on isolated rat hepatocytes and has been partially purified from rat liver using affinity chromatography. Isolated hepato- cytes exhibit approximately 30,000 ferritin binding sites/cell with a binding association constant (Ka) of 1 X 10' mol" liter. A binding assay has been developed which utilizes a hepatic ferritin receptor coupled to a microparticulate support to facilitate separation of bound and free ligand. This method yielded a K, of 3 x 10' mol" liter for the purified hepatic ferritin recep- tor. Binding of ferritin to the insolubilized receptor was partially inhibited by human lactoferrin but unaffected by 200-fold molar excess of bovine albumin, rat transferrin, or human asialoorosomucoid.

Ferritin is an ubiquitous iron storage protein found in all tissues of the body and there is a close correlation between body iron stores and plasma ferritin concentration (1,2). Injected ferritin has a plasma half life of 4-30 min and is taken up by the liver (3-7), specifically by hepatocytes (3). Use of the isolated perfused rat liver has indicated that the liver releases and takes up plasma ferritin, maintaining a perfusate ferritin concentration close to the plasma ferritin concentration, in uiuo, of the donor animal (8).
The precise mechanism by which ferritin is taken up by the liver is unknown, but the existence of a ferritin receptor has been suggested. This is supported by the finding that uptake of fluorescent-labeled ferritin by isolated rat hepatocytes occurs in a manner consistent with receptor-mediated endocytosis (9). The observations that after homogenization of whole liver some ferritin is membrane-associated (10) and in a detergent-solubilizable membrane fraction (11) further strengthen the concept that "hepatocytes have receptors for . . . ferritin" (12).
We have examined the binding of '"I-labeled rat liver ferritin to both isolated rat hepatocytes and a partially purified ferritin receptor preparation isolated from detergent-solubilized liver plasma membrane.

* This work was supported by the National Health and Medical
Research Council of Australia. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "aduertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
f National Health and Medical Research Council Research Officer.

MATERIALS AND METHODS
Male Sprague-Dawley rats weighing 250-350 g were used throughout. Bovine serum albumin and fetal calf serum were obtained from the Commonwealth Serum Laboratories (Australia). Collagenase was purchased from Worthington, Teric 12A9 from IC1 Chemicals (Sydney, Australia), human orosomucoid from Calbiochem-Behring, soybean trypsin inhibitor and agarose-coupled neuraminidase Type X-A from Sigma, and CNBr-activated Sepharose 4B from Pharmacia Fine Chemicals (Uppsala, Sweden). Matrex pel 102 was purchased from Amicon (Lexington, MA), Bolton and Hunter Reagent (NEX 120) from New England Nuclear, and Sac-cel (donkey anti-rabbit whole serum coupled to cellulose) from Wellcome Reagents (Sydney, Australia). All other chemicals were of reagent grade.
Human lactoferrin (13), rat transferrin (14), ferritin, and purified antibody to rat liver ferritin (7) were prepared and rat serum ferritin was assayed as previously described (8). The equilibrium binding constant ( K O ) of the rabbit antibodies to rat liver ferritin was 1.2 X los mol" liter. Human asialoorosomucoid was prepared by incubating 10 mg of orosomucoid with 1 unit of insoluble neuraminidase in 0.1 M acetate, pH 5.0, (10 ml) at 37 "C for 24 h. Asialoorosomucoid was purified by centrifugation at 11, OOO X g for 10 min to remove the insoluble neuraminidase followed by chromatography on a Sephadex G-25M column (1.5 X 8 cm, Pharmacia PDlO column) eluting with 0.145 M NaC1.
Rat liver ferritin (10 mg) or affinity purified rabbit antibody to rat liver ferritin (5 mg) in 0.1 M borate, pH 8.0, containing 0.5 M NaCl was coupled to 5 g of CNBr-activated Sepharose 4B at 4 "C for 18 h. Reactive groups in the affinity gel were blocked by addition of 0.1 M ethanolamine, pH 8.0, followed by stirring at 4 "C for 2 h and extensive alternate washing with 0.1 M acetate, pH 4.0, containing 0.5 M NaC1, and 0.1 M borate, pH 8.0, containing 0.5 M NaC1. 100 pg of rat liver ferritin was radiolabeled with "' 1 using 0.5 mCi of Bolton and Hunter Reagent (15). Labeled ferritin and free Iz5I were separated by chromatography on a Sephadex G-25M column (1.5 X 8 cm, Pharmacia PDlO), eluting with 20 mM KPO,, pH 7.4, containing 100 mM NaCl.
Protein concentration was determined by the deoxycholic acid/ trichloroacetic acid precipitation method of Peterson (16).
Preparation of fsolated Rat Hepatocytes-Isolated rat hepatocytes were prepared by a modification of the collagenase perfusion method of Berry and Friend (17) as adapted by Seglen (18). Singlepass perfusion was commenced in situ via the portal vein with a calcium-free Krebs-Ringer bicarbonate buffer, containing 20 mM Dglucose and gassed with 95% 0,,5% CO, (KRB). The liver was rapidly removed to a cabinet maintained at 37 "C (19) and perfusion was continued for a total of 10 min before commencing recirculating perfusion with KRB containing 5 mM CaCL, 0.01% soybean trypsin inhibitor, and 0.05% collagenase for a further 20 min. out into oxygenated MEM' at 37 "C. The resulting cell suspension The liver capsule was removed and the cells were carefully teased was filtered sequentially through a single layer of gauze followed by 100-pm Nylon mesh and allowed to settle at room temperature for 10 min. The supernatant was removed, the cells were resuspended in warm MEM, and the settling process repeated twice more. This yielded 200 X lo6 to 700 X lo6 cells with a viability of 85-95% as assessed by trypan blue exclusion.
Assay for Binding of 12'Z-Ferritin to Hepatocytes-Equilibrium binding studies were carried out at 4 "C. 1 X 10" hepatocytes were added to pre-chilled tubes containing MEM, varying amounts of ferritin (trace labeled with 1 pg of "'I-labeled ferritin), and 10% FCS in a final volume of 1 ml. The tubes were gently swirled at 4 "C for 2 h. The cells were then separated from the incubation medium by vacuum fdtration onto Whatman GF/A filter discs previously moistened with MEM, 10% FCS followed by washing with 5 X 2 ml Of icecold MEM. The fdter discs were then dried and bound '"I-labeled ferritin was determined on a Kontron MR252 y counter.

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Receptor for Ferritin 4673
Purification of Rat Liver Ferritin Receptor-Fresh rat livers weighing a total of 150 g were homogenized in 300 ml of 20 mM sodium phosphate, pH 7.4, containing 0.1 M NaCl and 1 mM phenylmethylsulfonyl fluoride in a Waring Blendor for 120 s. The homogenate was centrifuged at 30,000 g for 30 min and the supernatant discarded. The pellet was resuspended in 300 ml of PBS/PMSF and recentrifuged twice.
The pellet was then suspended in 200 ml of PBS/PMSF and solid Teric 12A9 was added to a r i a l concentration of 1% (w/v). The membrane preparation was solubilized by sonication (MSE ultrasonic homogenizer, 8 mm probe) for 5 min at 0 "C, fdtered through a layer of gauze, and then centrifuged at 30,000 X g for 20 min and the pellet discarded.
Solid ammonium sulfate (31.5 g/100 ml) was added with stirring followed by centrifugation at 10,000 X g for 20 min. The supernatant was carefully removed from under the floating cap of precipitate and the precipitate was dissolved in 0.1 M borate, pH 8.0, containing 0.5 M NaCl and 1% Teric 12A9 (B8BT). After dialysis against the same buffer a t 4 "C for 24 h, the preparation was centrifuged at 30,000 X g for 20 min.
The supernatant was applied to a column (1.6 X 20 cm) containing equilibrated with B8BT at 4 "C. 200 ml of B8BT were applied and 17 ml of anti-rat liver ferritin antibody coupled to Sepharose 4B the ferritin-binding protein fraction was then eluted with 100 ml of 20 mM glycine, NaOH, pH 10, containing 0.5 M NaCl and 0.5% Teric 12A9 (elution buffer). The column was then regenerated for further use by elution of bound ferritin with 100 ml of 10 mM glycine, HCI, pH 2, containing 150 mM NaCl, followed by 200 ml of B8BT. The ferritin-binding protein fraction was precipitated by the addition of solid ammonium sulfate (31.5 g/100 ml), stirred for 20 min, and centrifuged at 10,OOO X g for 20 min. The precipitate was resuspended in B8BT and dialyzed.
The dialyzed ferritin-binding protein fraction was then applied to a column (1.6 X 20 cm) containing 15 ml of rat liver ferritin Sepbarose 4B equilibrated with B8BT. After washing with 200 ml of B8BT, the ferritin receptor protein was eluted at pH 10 using 100 ml of the above elution buffer. The column was then re-equilibrated with B8BT.
The ferritin receptor fraction was concentrated by ammonium sulfate precipitation and dialyzed against B8BT as described above. Possible contamination by rabbit immunoglobulins, e.g. an antibody to rat liver ferritin was excluded by incubating the receptor preparation with donkey anti-rabbit whole serum coupled to cellulose at 4 "C for 18 h followed by removal of the antibody-cellulose complex by centrifugation at 2000 X g for 10 min.
Preparation of Insolubilized Receptor-The dialyzed ferritin receptor preparation containing 20-50 pg/ml of protein was added to Matrex pel 102 (2 ml of suspension (0.13 g)/10 ml of receptor solution) which had been washed with B8BT. After stirring the preparation for 18 h at 4 "C, 1 M ethanolamine, pH 8, (final concentration 0.1 M) was added and stirring was continued for 4 h. The resulting suspension was washed extensively with B8BT by centrifugation and resuspension in fresh buffer. The suspension was made up to a volume of 75 ml/g of Matrex pel with B8BT before use and was stored at 4 "C.
Assay of Binding of '"I-Ferritin to Insolubilized Ferritin Receptor-Polypropylene 1.5-ml microcentrifuge tubes containing 0.096 ml of PBS/PMSF plus ferritin (0.2-50 pg/ml) or other protein, 0.004 ml of '251-labeled ferritin (200 ng), 0.8 ml of B8BT, and 0.1 ml of insolubilized receptor suspension in B8BT were allowed to stand at 4 "C for 18 h. The assay tubes were then centrifuged at 11,000 X g for 2 min (Hettich Microliter centrifuge) and the supernatant carefully removed. The pellet was resuspended in 1 ml of ice-cold BSBT, recentrifuged, and the supernatant again removed. The radioactive content of the pellet was then determined on a Kontron MR252 y counter.

RESULTS AND DISCUSSION
The binding of lz5I-ferritin to isolated rat hepatocytes was essentially complete after 2 h of incubation. While a high level of nonspecific binding was observed (up to 60% of total binding) with hepatocytes, specific binding was nonetheless demonstrated (Fig. 1) and Scatchard plots (20) of the specific binding yielded a binding association constant ( K , ) of 1.1 * 0.4 X 10' mol" liter (mean -+ S.D.) with 3.2 k 1.6 X lo4 binding sites/hepatocyte (Fig. 2). Experiments were difficult to repeat in rats with normal or increased iron stores and reproducible results were obtained largely with hepatocytes from rats with relatively low iron stores (Serum ferritin concentration 30-150 The purification of the ferritin receptor was undertaken in an attempt to overcome the high nonspecific binding encountered using isolated hepatocytes. The preparation was based Binding of ferritin to the insolubilized receptor was maximal between pH 7.5 and 8.0 and was greatly reduced below pH 6 and above pH 9. Ferritin-anti-ferritin complexes on the other hand have been shown to be unstable below pH 6.0 but stable above pH 9.0. Therefore, advantage was taken of this relative stability of the anti-ferritin-ferritin complex and elution of the receptor from ferritin after binding to the anti-ferritin column was accomplished at pH 10. Contamination of the receptor preparation with rabbit antibodies to rat liver ferritin was excluded by incubation of the receptor preparation with donkey anti-rabbit whole serum coupled to cellulose. Antiferritin antibodies were not detectable after the second affinity chromatography step on ferritin-Sepharose. Assay of the binding of 1251-ferritin to the receptor using methods of separating bound and free ligand such as ammonium sulfate and polyethylene glycol precipitation proved difficult because of co-precipitation of free and bound ferritin. Similarly, there was insufficient difference in either elution volume or density of the ferritin-receptor complex as compared with ferritin to facilitate separation of bound and free ferritin by gel chromatography or centrifugation. However, ng/ml). FIG. 1 (left). Binding of 1 pg of lZ5Ilabeled rat liver ferritin at 4 "C/2 h to 1 X 10' isolated rat hepatocytes in MEM, 10% FCS in the presence of an increasing Concentration of unlabeled ferritin. FIG. 2 (right). Scatchard plot of the binding data derived from the experiment presented in Fig. 1. K , sites/hepatocyte.  3 (left). Binding of 200 ng of '261-labeled rat liver ferritin at 4 "C/ 18 h to insolubilized ferritin receptor in the presence of an increasing concentration of unlabeled ferritin. FIG. 4 (right). Scatchard plot of the binding data derived from the experiment presented in Fig. 3. K, = 3 X 10' mol" liter. coupling of the partidy purified ferritin receptor to a micropellicular support resulted in an insolubilized receptor which allowed effective separation of bound and free ferritin by centrifugation.
The binding of '251-labeled rat liver ferritin to the insolubilized receptor in the presence of increasing amounts of unlabeled ferritin after 18-h incubation is shown in Fig. 3. From Scatchard plots of the binding of ferritin to the insolubilized receptor ( Fig. 4) a K , of 2.7 & 0.7 X 10' mol" liter was derived, which is in agreement with the K,, obtained in the hepatocyte binding studies. This provides additional evidence that the receptor is present on hepatocytes (3,8).
Rat liver ferritin has been shown to contain approximately 2.5% carbohydrate (22). This carbohydrate consists of mannose, glucose, galactose, glucosamine, and galactosamine but sialic acid and fucose were not detectable (22). Binding specificity studies, therefore, were carried out to determine whether the characteristics of the ferritin-binding protein resembled the previously described asialoglycoprotein receptor (23, 24), the transferrin receptor (25), or the glycoprotein receptor which has been described on macrophages (24, 26-28). The asialoglycoprotein receptor shows specificity for galactose-or glucose-terminated glycoproteins such as human asialoorosomucoid (23, 24), while the glycoprotein receptor which has been previously described on macrophages is specific for fucose-, mannose-, and N-acetylglucosamine-terminated glycoproteins (24, 26-28) including lactoferrin. Binding studies were carried out using bovine serum albumin (a nonspecific protein), rat transferrin, AsOR, or human lactoferrin in the assay system. The binding of ferritin was unaffected in the presence of a greater than 200-fold molar excess of bovine serum albumin, AsOR, or transferrin (Fig. 5), indicating that the hapatocyte ferritin receptor differs from both the asialoglycoprotein and transferrin receptors. Human lactoferrin showed some degree of competition with ferritin binding to the ferritin receptor (Fig. 6), however, the binding affinity of ferritin to the receptor was 5-fold higher than the binding of lactoferrin. This competition may reflect the presence of a common carbohydrate moiety on both the ferritin (22) and the lactoferrin molecules (29).
Accurate quantification of the total amount of hepatic receptor was not possible in these experiments because of the difficulty in estimating recoveries at various stages of the purification. However, calculation of the total binding capacities in intact hepatocytes and in the final insolubilized receptor preparation indicated an overall recovery of 515%. Preliminary SDS electrophoresis of the receptor has shown three faint bands with an M , of 30,000-50,000, but more complete characterization must await the preparation of larger quantities of the binding protein.
The presence of a ferritin receptor has been reported previously on guinea pig reticulocytes (30). Pollack   in this study have reported a dissociation constant for the binding of ferritin to reticulocytes (&) of 0.3 x lo-'" M (or a K , of 2 X lo7 mol" liter, if a correction is applied to allow for the incubation volume of 1.45 ml and where K , = l/K<z). However, the large difference observed between binding at 37 and 0 "C suggests in these studies that internalization of bound ferritin by reticulocytes occurs at 37 "C. In the presence of internalization and in the short time period (20 min) used for incubation, an equilibrium cannot be assumed, and analysis by Scatchard plot and derivation of binding equilibrium constants may well be inappropriate.
We have demonstrated a hepatic ferritin receptor in rats by the use of an insolubilized receptor preparation which permitted analysis of the binding characteristics of the receptor. The technique should be generally applicable to the analysis of ligand-receptor interactions. While the precise physiological role of the ferritin receptor is not yet understood, we have presented evidence for the existence of a specific mechanism responsible for the rapid uptake of ferritin from the circulation (8).