Binding and Endocytosis of Apo- and Holo-Lactoferrin by ... · urated (holo-Lf) lactoferrin were used. At 4 “Cy cells bound ‘““I-apo-Lf and “”I-holo-Lf with nearly identi-

Binding and Endocytosis of Apo- and Holo-Lactoferrin by Isolated Rat Hepatocytes*

(Received for publication, July 19, 1991)

Douglas D. McAbeeS and Kari Esbensen From the Department of Biological Sciences, The University of Notre Dame, Notre Dame, Indiana 46556

We characterized binding and endocytosis of ‘“I- bovine lactoferrin by isolated rat hepatocytes. Iron- depleted (apo-Lf), -30% saturated (Lf), and iron-saturated (holo-Lf) lactoferrin were used. At 4 “Cy cells bound ‘““I-apo-Lf and “”I-holo-Lf with nearly identical apparent first order kinetics (t1/< = -42 min). Holo- Lf and apo-Lf competed with each other for binding. Hepatocytes bound lactoferrin optimally at pH 2 7 but poorly at pH I 6. Ca”+ (2100 p ~ ) enhanced Lf binding to cells, and holo-Lf remained monomeric with Ca“+ present as determined by gel filtration chromatography. With Ca”+, cells exhibited -10” high affinity sites (& -20 nM) and -io7 low affinity sites (& - 700 nM) for both apo- and holo-Lf. Without Ca“+, cells bound ‘”“I-holo-Lf by the low affinity component only. EGTA and dextran sulfate together released 2 90% ‘””I-Lf prebound at 4 “Cy but individually removed separate populations of surface-bound ’”“I-Lf. Cells bound ““I- Lf in a Ca”+-dependent manner with dextran sulfate present. We conclude that the high affinity but not the low affinity sites require Ca”+; only the low affinity sites are dextran sulfate-sensitive. Neither transferrin nor asialo-orosomucoid blocked lactoferrin binding to hepatocytes. Some cationic proteins but not others inhibited lactoferrin binding. At 37 O C , hepatocytes endocytosed ‘““I-apo-Lf and ’““I-holo-Lf similarly, and hyperosmolality (>500 mmol/kg) blocked uptake by -90%. These data support the proposal that hepatocytes regulate blood lactoferrin concentration by receptor-mediated endocytosis.

Lactoferrin belongs to the transferrin family of non-heme iron-binding glycoproteins. A single-chain bilobed protein (80 kDa), Lf’ binds reversibly two Fei+, contains two N-linked complex biantennate oligosaccharides, and has a PI = 8.2 (for review, see Baker et al., 1987). Virtually all body fluids contain Lf (Bennett and Kokocinski, 1979; Masson et al., 1966). Blood Lf originates in polymorphonuclear leukocytes which release

* A portion of this work was supported by a Jesse H. Jones Faculty Research Grant (to D. D. M.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

4 To whom correspondence should be addressed. The abbreviations used are: Lf, lactoferrin; AMPSO, 3-[(1,1-

dimethyl-2-hydroxyethyl)amino]-2-hydroxypropanesulfonic acid; ASOR, asialo-orosomucoid BME, basal medium Eagle’s; BES, N,N- bis(2-hydroxyethyl)-2-aminoethanesulfonic acid BSA, bovine serum albumin; GM-CSF, granulocyte macrophage-colony stimulating fac- tor; HBSS, Hanks’ buffered salt solution; HEPES, N-2-hydroxyethyl- piperazine-N’-2-ethanesulfonic acid HBS, HEPES-buffered saline; MES, 2-(N-morpholino)ethanesulfonic acid HEPPS, 442-hydroxyethyl)-1-piperazinepropanesulfonic acid; EGTA, [ethylenebis(oxy- ethylenenitrilo)]tetraacetic acid SDS, sodium dodecyl sulfate.

Lf during exocytosis of specific granules. Lf concentrations in the blood normally remain low (-20 nM), because the liver rapidly clears Lf from the circulation (Bennett and Kokocin- ski, 1979; Imber and Pizzo, 1983), amounting to 15-25 mg of Lf daily in humans (Bennett and Kokocinski, 1979).

Lf in blood apparently possesses at least two functions. First, Lf inhibits division of macrophage and granulocyte progenitor cells in bone marrow (Hangoc et al., 1987; Zucali et al., 1989). It is thought that blood Lf, in concert with GM- CSF, regulates the production of white blood cells by blocking GM-CSF release from macrophages and fibroblasts (Zucali et al., 1989). Lf inhibits GM-CSF release at picomolar concentrations, and inhibition is proportionate to Lfs iron content (Broxmeyer et al., 1978). Second, blood Lf may regulate iron retrieval and processing during erythrocyte catabolism and biogenesis. Reticuloendothelial cells degrading senescent erythrocytes release iron slowly into the circulation. It has been proposed that Lf competes with transferrin for iron discharged by reticuloendothelial cells. Hepatic cells ingest iron-Lf complexes and store the liberated iron with ferritin (Retegui et al., 1984). In this way, blood Lf, present largely in the apo form, diverts iron from a “fast” recycling pool to a “slow” recycling pool, thereby regulating the amount of iron available for erythrocyte biogenesis. Accordingly, iron di- verted to the slow pathway increases during systemic trauma or infection possibly because plasma Lf levels increase under these conditions (Peterson et al., 1988; Van Snick et al., 1974). The liver’s ability to clear blood Lf, therefore, is very important because the extent of Lf s effects on these phenomena is proportional to its concentration in blood. Elevation of plasma Lf provokes severe neutropenia (Boxer et al., 1982), inhibits hepatic uptake of chylomicron remnants (Huettinger et al., 1988), and may mediate the anemia of chronic disease (Lee, 1983). Thus, continuous clearance of plasma Lf constitutes a significant hepatic homeostatic function.

I t was initially proposed (Prieels et al., 1978) that the liver cleared Lf via the hepatic fucosyl receptor (Lehrman and Hill, 1986; Townsend and Stahl, 1981). Subsequent studies showed, however, that clearance of blood Lf did not require hepatic recognition of Lf s carbohydrates. Glycoconjugates bearing fucose, galactose, N-acetylglucosamine, or mannose did not inhibit hepatic accumulation of human Lf from mouse circulation (Imber and Pizzo, 1983). Moreover, human granulocyte Lf is not fucosylated (Derisbourg et al., 1990). Lfs polypep- tide, but not its carbohydrate, determined its hepatic clearance from the circulation (Moguilevsky et al., 1984), and Lfs cell binding domain may reside within its N-terminal 100 amino acids (Rochard et al., 1989). Hepatocytes, Kupffer cells, and liver endothelial cells can accumulate Lf from the circulation (Courtoy et al., 1984; Imber and Pizzo, 1983; Regoeczi et al., 1985). It is unknown whether the liver clears Lf via receptor-mediated endocytosis or by adsorptive pinocytosis. Putative Lf receptors have been identified in phytohemagglu-

23624

Hepatocyte Binding/Endocytosis of Lactoferrin 23625

tinin-stimulated peripheral blood lymphocytes (Mazurier et al., 1989), in intestinal mucosal epithelia (Hu et al., 1990), and on the surfaces of Haemophilus influenzae (Schryvers and Morris, 1988) and Neisseria meningitidis (Schryvers, 1989). As yet, an hepatic Lf receptor has not been identified. In addition, the biologic effects of Lf on hepatic cell function are completely unknown.

A crucial step in understanding the biology of Lf is to examine its interaction with cells. To this end, we performed experiments examining Lf binding to and uptake by isolated rat hepatocytes. This study constitutes the first examination of Lf interaction with intact isolated hepatic cells. We found that (i) hepatocytes bound bovine Lf via two populations of sites: Cay+-dependent high affinity sites and Ca"-independent low affinity sites; (ii) binding was optimal a t neutral to basic pH; (iii) Lf bound hepatocytes regardless of its iron content; and (iv) hepatocytes internalized bound Lf via a clathrin-dependent endocytic pathway.

EXPERIMENTAL PROCEDURES

Materials-Bovine colostrum Lf, bovine transferrin, human orosomucoid, BSA (Fraction V), dextran sulfate (5000 Da), lysozyme (chicken egg white, EC 3.2.1.17), lactoperoxidase (bovine .milk, EC 1.11.1.7), cytochrome c (beef heart, type V-A), protamine sulfate (salmon, grade X), AMPSO, BES, MES, and neuraminidase (type X) were purchased from Sigma. Collagenase (Type A and D) was obtained from Boehringer Mannheim. BSA was also obtained from Gibco. HEPES and HEPPS were obtained from Fisher Biochemicals. 1,3,4,6-Tetrachloro-3a,6a-diphenylglycoluril (Iodogen) and chromatography molecular weight standards (Mol-Ranger) were from Pierce Chemical Co. Na'"'1 (-17 Ci/mg iodine) was from ICN Biomedicals, Inc. (Irvine, CA). All other chemicals were reagent grade. HBSS was obtained from GIBCO. HBS contained 150 mM NaCI, 3 mM KCI, 10 mM HEPES, pH 7.4. BME (GIBCO) was supplemented with 2.4 g/ liter HEPES, pH 7.4, and 0.22 g/liter NaHCO:,. BME/BSA is BME containing 0.1% (w/v) BSA. ASOR was prepared from human orosomucoid (a-acid glycoprotein) by desialylation with neuraminidase as described elsewhere (Schachter et al., 1970; Weigel and Oka, 1982).

Hepatocytes-Male Sprague-Dawley rats (150-300 g; Harlan Lab- oratories, Indianapolis, IN) were fed standard laboratory chow and water ad libitum. Hepatocytes were prepared by a modification of a collagenase perfusion procedure (Seglen, 1973) as described previously (Oka and Weigel, 1987). Cells were kept a t -30 "C during the filtration and differential centrifugation steps. Final cell pellets suspended in ice-cold BME/BSA were 2 85% viable and single cells. Before experiments cell suspensions (2-4 X loG cells/ml in BME/ RSA, 10% of the flask volume) were incubated at 37 "C for 60 min to allow recovery from the isolation procedure. Viability was determined microscopically by trypan blue exclusion. Lf and Transferrin Preparation-Bovine colostrum Lf (commercial

preparation >90% pure electrophoretically) at 5 mg/ml in 100 mM KCI, 10 mM KH2P04, pH 7.2, was chromatographed a t 4 "C in the same buffer over either DEAE-Sephadex A-50 or DE32 to remove contaminating immunoglobulin A (Hashizume et al., 1987). Lf was depleted of iron (apo-Lf) by dialysis against citric acid as described elsewhere (Masson and Heremans, 1968). Lf was saturated with iron (holo-Lf) by dialysis against sodium citrate-supplemented FeCI:, as described elsewhere (Schryvers, 1988). Transferrin was iron saturated as described elsewhere (Klausner et al., 1983), then chromatographed on a desalting column (Pierce GF-5 column) equilibrated with HBS. Purified Lf and transferrin were dialyzed against HBS, then filter- sterilized (0.2 pm) and stored a t -20 "C prior to use. The amount of iron bound by Lf and transferrin was estimated from the A,,,s ,,,,> ratio, which was > 0.04, consistent with > 85% saturation (Hash- izume et al., 1987). '""IApo- and ]""I-holo-Lf, prepared by the method of Fraker and Speck (Fraker and Speck, 1978), had specific activities of 20-130 dpm/fmol.

Homogeneity of labeled and unlabeled apo- and holo-Lf used for all binding studies was determined by SDS-polyacrylamide gel electrophoresis (Fig. 1). Lf was electrophoresed on 7.5% Laemmli gels (Laemmli, 1970) containing 0.1% SDS under nonreducing conditions using a Mini-PROTEAN I1 slab gel apparatus (Bio-Rad), then detected either by silver stain (Merril et al., 1980) or by autoradiography after 16 h exposure to Kodak X-Omat-AR film. Purified bovine Lf

A B

- -116 \ - 92 \

-0 - 67 \

-45.

1 2 -

1 2

FIG. 1. SDS-polyacrylamide gel electrophoresis and auto- radiographic analysis of '"I-apo-Lf and ""I-holo-Lf. Purified unlabeled or iodinated apo- and holo-Lf (-1 pg/lane) were electrophoresed as described under "Experimental Procedures." Panel A, silver stain of apo-Lf (lane 1 ) and holo-Lf (lane 2). Panel B, autoradiography of "..'I-apo-Lf (lane 1 ) and "..'I-holo-Lf (lane 2). Relative migration of molecular weight markers is designated in kilodaltons. Higher molecular weight bands present in some lanes were oligomeric forms of Lf generated artifactually during sample preparation for electrophoresis. This was determined as follows. Lf was electrophoresed preparatively, then transferred to nitrocellulose paper. The major Lf protein band a t 78 kDa was excised and extracted from the paper by heating a t 80 "C in Laemmli sample buffer. These higher molecular weight forms with M, corresponding to Lf dimers and trimers were observed when recovered Lf was re-electrophoresed and detected by silver stain. These oligomers routinely constituted <3% of the total electrophoresed Lf as determined by laser scanning densitometry.

migrated routinely either as a single band with an apparent molecular mass of 78 kDa or as a tight doublet between 76-78 kDa. Apo- and holo-Lf (unlabeled and iodinated) electrophoresed similarly, and neither ""I-apo-Lf nor "..'I-holo-Lf underwent significant radiolysis during several weeks of storage at 4 "C.

Binding Assays-Unless otherwise stated, cells were assayed for Lf binding in the following manner. After 37 "C equilibration, cells were chilled on ice and washed in either HBSS or HBS at 4 "C prior to binding assays. Routinely, -10" cells were incubated with "'I-Lf (0.5- 1.0 pg/ml) in 0.5 ml of HBSS or HBS supplemented with 5 mM CaCI? and 5 mM MgCl? for 90 min on ice. Nonspecific binding was performed by including 100-500 fold molar excess of unlabeled Lf in the binding mixture. The cells were washed twice by centrifugation in ice-cold binding buffer, suspended in 1.0 ml ice-cold binding buffer then transferred to clean plastic tubes. The samples were supplemented with 0.1 ml of 1 N NaOH, heated 15-20 min at 37 "C, then assayed for radioactivity and protein.

General Procedures-Protein was determined by the bicinchoninic acid protein assay procedure (Smith et al., 1985) (Pierce Chemical Co.) using BSA as standard. Hepatocytes contain 1.1 mg of protein/

HlOOOB rotor) for 2 min at 4 "C in a Sorvall RT6000B table-top 10" cells. Centrifugation of cell suspensions was at 800 rpm (Sorvall

centrifuge (Du Pont). ".*'I radioactivity was determined using a Pack- ard Cobra Auto-Gamma Counting System Model 5002 (Packard Instrument Co., Downers Grove, IL). Osmolality was determined using a Wescor 5500 vapor pressure osmometer (Wescor, Inc., Logan, UT). Spectral analysis of Lf and transferrin for iron content was performed using a Beckman DU-64 spectrophotometer.

RESULTS

Lf Binding to Hepatocytes at 4 "C-We examined the kinetics of ""I-apo-Lf and '"'I-holo-Lf binding to isolated hepatocytes a t 4 "C. If Lf binding to hepatocytes is iron-dependent, then the liver may accumulate holo-Lf but not apo-Lf from the circulation. This is relevant because transferrin shares -50% sequence homology with Lf (Baker et al., 1987) and binds cells with high affinity only when it is saturated with iron (Dautry-Varsat et al., 1983; Klausner et al., 1983). As seen in Fig. 2 A , hepatocytes bound both "'I-apo- and "'I- holo-Lf in a time-dependent manner, reaching equilibrium after -90 min. Nonspecific binding for both types of Lf was

23626 Hepatocyte BindinglEndocytosis of Lactoferrin

0 30 60 90 120 150 180

Time (min) 5 . 5 . I . I . I . I .

3 . 0 r ' I ' I ' " " ' -

0 20 40 60 80 100

Time (min)

FIG. 2. Kinetics of lactoferrin binding to hepatocytes. A , cells were incubated a t 4 "C with I2'I-apo-Lf or "'I-holo-Lf in HBS containing divalent cations for the designated times, then assayed for cell-associated radioactivity and protein as described under "Experi- mental Procedures." Experiments using the two different Lf forms were done with cells isolated on different days. Samples for the two binding experiments each contained -lo6 cells. B, apparent first order association constants were determined by calculating the slope of In Lf bound as a function time. Half-times for Lf binding were calculated using the equation ( k J ( 6 J = In 2. Apo-Lf: k, = 0.016 rnin", r = 0.98, t1,> = 43 min; holo-Lf: k. = 0.017 rnin", r = 0.92, tI,' = 41 min. Symbols represent the mean of duplicate samples that differed by 5 10%.

TABLE I Effect of divalent cations on lactoferrin binding to hepatocytes

Cells were washed in the appropriate binding buffer at 4 "C prior to binding assays. "'I-Lf used had physiologic iron load (-30% saturation). Binding buffers contained HBS either alone (no additions) or supplemented with the designated components. Binding mixes contained 0.5 pg/ml "'I-Lf (0.5 ml). Cells were assayed for binding as described under "Experimental Procedures." Nonspecific binding was assessed by performing binding assays in 500-fold excess of unlabeled Lf. Values represent means of duplicate samples k standard deviation. Specific binding = (total binding) - (nonspecific binding).

Binding buffer '2sII-Lf bound

Total Nonspecific Specific frnol/rng protein

No additions 540 f. 7 209 * 7 331 10 mM CaC12 896 2 132 123 2 14 773

10 mM MgC1, 298 k 54 61 k 4 233 10 mM EGTA 462 f. 14 194 ? 18 268

< 15%. The apparent first order k , of apo-Lf and holo-Lf (Fig. 2B) were 0.016 min" and 0.017 rnin", respectively, suggesting that the rate of Lf binding to cells was essentially independent of Lf s iron content.

To examine divalent cation requirements for hepatocyte binding of Lf, we incubated cells with '"I-Lf at 4 "C in HBS either alone or with EGTA, CaC12 or MgC1, (Table I). Hepa- tocytes bound ""I-Lf in the absence of divalent cations, and EGTA reduced '"I-Lf binding only slightly compared to HBS alone. In each of these cases, nonspecific binding was -40% of total. In contrast, cells incubated in Ca"-containing HBS bound -65% more "'I-Lf than did cells without Ca2+; nonspecific binding was < 20% of total. Mg" did not increase Lf binding to cells but reduced nonspecific binding. Thus, he-

patocytes did not require divalent cations to bind Lf but Ca2+ increased Lf specific binding. Therefore, we measured Lf binding as a function of Ca2+ concentration (Fig. 3). Hepato- cytes bound similar amounts of "'I-apo-Lf and I2'II-holo-Lf in the absence of Ca'+. Between 80 and 1000 WM CaCl,, binding of both ligands approximately doubled. CaC12 21 mM did not stimulate Lf binding further. Cells bound "'I-apo-Lf and I2'I-

holo-Lf to similar extents over most of the CaC1, concentration range, although at CaCl, 2 200 I.LM cells bound slightly more '"I-holo-Lf than "'I-apo-Lf. Thus, these data suggested that hepatocytes possess both Ca2+-independent and Ca2+- dependent Lf binding sites.

In the presence of millimolar CaCl,, human Lf can form tetramers detected by gel filtration chromatography and equilibrium density centrifugation (Bennett et al., 1981). Such oligomerization might account for the Ca2+-dependent increase of Lf binding to hepatocytes. To determine the effect of Ca'+ on oligomeric state of bovine Lf, holo-Lf was chromatographed on Sephacryl S-300 equilibrated in either CaC1, or EGTA (Fig. 4). Under these conditions, bovine Lf migrated as a monomer regardless of the presence of Ca2+. These data suggest that the Ca2+-enhanced binding of Lf by hepatocytes was not due to binding of Lf oligomers formed in Ca2+- containing medium.

The equilibrium binding constants for Lf were determined by incubating '"'I-apo-Lf (Fig. 5A) or 12'I-holo-Lf (Fig. 5B)

0- 0.01 0.1 1 .o 10

CaCI2 (rnM)

FIG. 3. Lf binding to hepatocytes as a function of CaC12. Equilibrated cells were washed four times with cold HBS in the absence of divalent cations, then assayed at 4 "C for binding of "'1- apo-Lf or "'I-holo-Lf (1 pg/ml) as described under "Experimental Procedures" in the absence or presence of different amounts of CaC1,. Symbols reflect the mean of duplicate samples that differed by 5 10%.

18 22 26 30 34 38 42

Fraction FIG. 4. Gel filtration chromatography of Lf: effect of CaC12.

Holo-Lf (8 mg/ml) was dialyzed exhaustively against HBS containing either 5 mM CaC12 and 5 mM MgC1, or 5 mM EGTA and 5 mM MgCl,, then chromatographed downward on a Sephacryl S-300 column (80 X 1.5 cm) equilibrated in the same buffer. The volume of Lf added was 1.0 ml, and column fractions contained 2.0 ml. Fractions were assayed for absorbance at 280 nm. The column's void volume (arrow) was determined by chromatography of blue dextran (Mr> 2 million). Molecular weight standards included beef heart catalase (240,000), rabbit muscle aldolase (158,000), and bovine albumin (67,000).

Hepatocyte BindinglEndocytosis of Lactoferrin 23627

~ p o - L t Bound (pmol/mg protein)

B

0.000 ' 1 ' ; ' h? 2 0

Holo-Lf Bound (pmol/mg protein) 0.12

5 0

C a 0.06 300 600 903

\ X

0 . 0 2

C 0 " ' " " "

0 4 1 2 16 2 0

Holo-Lt Bound (prnohg protein) FIG. 5. Equilibrium binding of Lf to hepatocytes. A, equili-

brated cells (lo6 cells/0.5 ml) were incubated on ice with 0.3 pg/ml "'I-apo-Lf with designated amounts of unlabeled apo-Lf for 90 min on ice in HBS supplemented with 5 mM CaC1, and 5 mM MgCl,. The cells were centrifuged, and the amount of free "'I-apo-Lf in the supernatants was determined. The cells were then washed twice, and bound radioactivity and protein were determined. Equilibrium binding constants were determined by the method of Scatchard (Scat- chard, 1949) from the specific binding data (inset). Isotherms were calculated by linear regression: rhigh = -0.98; n o w = -0.99. B, equilibrated cells (106/0.5 ml) were incubated on ice with 0.8 pg/ ml of 12SI-holo-Lf with designated amounts of unlabeled holo-Lf for 90 min, assessed for free and bound "'I-holo-Lf, and analyzed for equilibrium binding constants as described in A. Isotherms were calculated by linear regression: rhigh = -0.98; rlow = -0.98. C, cells were assayed for '2'I-holo-Lf binding as described in B, except that cells were incubated with 1 pg/ml 12sI-holo-Lf in HBS containing 5 mM EGTA and 5 mM MgC1,. Equilibrium binding constants were determined as described in A. Isotherm was calculated by linear regression: r = -0.88. Binding constants calculated from these data are presented in the text.

with cells at 4 "C with excess unlabeled apo- or holo-Lf. Specific binding in these experiments, which were done in the presence of 5 mM CaC12, was 2 85% of total. In the presence

(Fig. 5, A and B, insets). Analyses of these binding data indicated that both apo-Lf and holo-Lf bound to hepatocytes by a high and low affinity component. As calculated from multiple equilibrium binding experiments, hepatocytes expressed 0.7 f 0.3 x 10" high affinity sites (& = 16.5 & 0.7 nM; n = 2) for '"I-apo-Lf and 1.3 f 0.3 X 10" high affinity sites (& = 28 f 19 nM; n = 3) for '"I-holo-Lf. Similarly, hepatocytes expressed 9.0 f 5.4 x lo6 low affinity sites (Kd = 520 f 57 nM; n = 2) for "'I-apo-Lf and 9.1 f 2.5 X lo6 low affinity sites (& = 903 f 680 nM; n = 3) for "'I-holo-Lf. Because binding constants for apo- and holo-Lf were very

of ca2+ , 1'" I-Lf binding began saturating at >IO0 pg Lf/ml

similar, it is likely that Lf binds to the same cell surface molecules regardless of its iron content. When incubated with "'I-holo-Lf in the absence of Ca2+, hepatocytes bound Lf by a low affinity component only (Fig. 5C). Under these conditions, hepatocytes bound > 100 million molecules of 12sI-holo- Lf at saturation (Kd 4.0 p ~ ) suggesting that high affinity but not low affinity hepatocyte Lf binding sites are Ca2+-dependent.

Specificity of Lf Binding to Hepatocytes-The specificity of Lf binding to hepatocytes was examined to determine what affects a variety of other ligands or charged molecules had on the cell binding of Lf. In the first experiment, hepatocytes were incubated with "'I-holo-Lf in the presence or absence of unlabeled Lf (apo or holo forms), holotransferrin, or ASOR. Because transferrin and Lf are very similar molecules, Lf could be binding hepatocyte transferrin receptors. We also tested ASORs ability to block cell binding of Lf because limited desialylation of Lf might account for the Ca"-dependent increase in 'T-Lf binding to cells by interaction with the galactosyl receptor (Wall et al., 1980; Weigel, 1980). As seen in Fig. 6, the amount of l2'1-holo-Lf bound by hepatocytes decreased 5 90% when incubated with 5 500 molar excess of unlabeled apo- or holo-Lf. Similar results were obtained if '2'I-apo-Lf was used instead of '251-holo-LfL. It appears, therefore, that apo-Lf and holo-Lf bind to the same sites on the surfaces of hepatocytes. In contrast, holo-transferrin and ASOR did not compete with 'ZsII-holo-Lf for binding to cells suggesting that neither transferrin nor galactosyl receptors on these cells bound Lf.

Because it is cationic, Lf bound by low affinity Ca2+- independent sites may result from nonspecific electrostatic interaction to hepatocyte surfaces. It was reported previously that strongly cationic proteins competed with human Lf for binding to isolated rat liver plasma membranes (DeBanne et al., 1985) suggesting that all Lf binding to hepatocyte membranes is through anionic sites. Lf, however, may bind intact cells with different specificity than isolated membranes. To test for this, we incubated cells at 4 "C with l2'1-holo-Lf with or without a 500-molar excess of unlabeled holo-Lf, protamine sulfate (PI 12.1; Felix, 1960), lactoperoxidase (PI 9.6; Polis and Shmukler, 1955), lysozyme (PI 11.4; Spector, 1956), or cytochrome c (PI 10.6; Spector, 1956) and assayed for 'T- holo-Lf bound to these cells. As seen in Fig. 7, unlabeled holo- Lf reduced "'I-holo-Lf binding by 84%, and protamine sulfate and lactoperoxidase also reduced "9-holo-Lf binding by 87 and 68%, respectively. On the other hand, lysozyme and

240 ~ . . . ~ . . . ~ . . . ~ . . ~ ~ . . . ~ . . . , ; ; i s o ; t I " 0

25 7 g 1 2 0 :

: o holo-Lf apo-Lf

2 E . 0" -

0 holo-Tf

T Z '

6 0 / * N u .-

O d ' ' >do' ' Zdo' ' '360' ' kdo' ' '560 ' '

Competitor Added (molar excess) FIG. 6. Holo-Lf binding to hepatocytes: effect of transferrin

and ASOR. After equilibration, cells (106/0.5 ml) were incubated for 90 min on ice with "'I-holo-Lf (0.8 pg/ml) with designated amounts of unlabeled holo-Lf, apo-Lf, holotransferrin, or ASOR. Samples were assayed for bound radioactivity and protein as described under "Ex- perimental Procedures." Each symbol represents the mean of duplicate samples that differed by 5 10%. Estimated molecular weights of ligands: holotransferrin, 78 kDa; asialo-orosomucoid, 40 kDa.

23628 Hepatocyte BindinglEndocytosis of Lactoferrin

0 60 120 180 240 '251-holo-Lf Bound (fmolisample)

FIG. 7. Binding of holo-Lf by hepatocytes: effect of basic proteins. Equilibrated cells (-106/0.5 ml) were incubated on ice for 90 min with "'I-bolo-Lf (0.8 pg/ml) in the absence (no additions) or presence of a 500 molar excess of unlabeled holo-Lf (0.4 mg/ml), protamine sulfate (25 pg/ml), lactoperoxidase (0.46 mg/ml), lysozyme (72 pg/ml), or cytochrome c (67 pg/ml), then assayed for bound radioactivity as described under "Experimental Procedures." Values represent the mean of triplicate samples, and bars reflect standard deviation.

200-

1 5 0 -

1 0 0 -

5 0 - E holo-Lf

-200

- 1 6 0

- 1 2 0

- 8 0

-40

0 ~ " " " ' " ' ' ~

pH

FIG. 8. Binding of Lf by hepatocytes: effect of pH. Cells were assayed for "'I-holo-Lf binding (1 pg/ml, 0.5 ml) and '"I-Lf (physiologic iron load, - 30% saturation; 0.5 pg/ml) at 4 "C as described under "Experimental Procedures." Each binding solution contained 0.15 M NaC1, 3 mM KC1, 5 mM MgC12, and 5 mM CaC12. Buffers for binding mixes were as follows: pH 5, 10 mM sodium acetate; pH 6, 10

AMPSO. Prior to binding assay, cells were washed with the appropriate binding buffer on ice. Values represent specifically bound Lf (total binding minus nonspecific binding). Symbols represent the mean of duplicate samples that differed by 5 10%. Experiments using the two forms of labeled Lf were performed on cells from different days.

4 5 6 7 8 9 1 ;

mM MES; pH 7 , l O mM BES; pH 8,lO mM HEPPS; pH 9,lO mM

cytochrome c reduced Lf binding by 5 10%. Thus, while some cationic molecules (protamine sulfate, lactoperoxidase) competed with Lf for binding to cells, other cationic proteins (lysozyme, cytochrome c) did not. I t appears, therefore, that the binding of Lf to hepatocytes is not due solely to an electrostatic interaction between Lf and acidic groups on hepatocyte surfaces.

pH Dependence of Lf Binding-Most Class I1 endocytic receptors bind ligand poorly or not a t all at pH 6.0 (Ander- son and Kaplan, 1983). It is thought that this pH sensitivity allows recycling endocytic receptors to disengage from their ligands in the slightly acidic environment of endosomes before migrating back to the cell surface. Accordingly, if Lf uptake by hepatocytes is receptor-mediated, then the interaction of Lf with cells may exhibit similar pH dependence on surface binding. To test for this, we assayed hepatocytes for "'I-Lf binding at 4 "C at pH 5-9 (Fig. 8). Cells bound '"I-Lf maximally at ? pH 7 regardless of L f s iron content. Nonspecific binding was 5 30% of total and remained fairly constant regardless of pH. At pH 5, specific binding diminished 65%.

These results indicate that hepatocytes bind Lf in a pH- dependent manner similar to that observed for other ligands which bind specific surface receptors (Anderson and Kaplan, 1983).

Removal of Lf from Hepatocyte Surfaces-As a requirement for biochemical measurement of "'I-Lf uptake by cells, conditions were determined that removed surface-bound "'I-Lf without disrupting the cells. Such conditions could be used subsequently to distinguish endocytosed 'T-Lf from total bound "'I-Lf (surface and intracellular). Because hepatocytes require Ca2+ to bind Lf to high affinity binding sites, washing cells in EGTA-containing medium should remove this bound Lf. Polyanions (heparin, dextran sulfate) remove low density lipoprotein particles bound to surfaces of several types of cultured cells apparently by enhancing the dissociation rate of low density lipoprotein receptor/low density lipoprotein particle complexes (Goldstein et al., 1983). Fucoidin prevents Lf interaction with liver by complexing directly with Lf (Im- ber and Pizzo, 1983), and dextran sulfate inhibits hepatic clearance of human holo-Lf from mouse circulation when co- injected with the Lf (Retegui et al., 1984). Thus, we tested dextran sulfate's ability to remove "'I-Lf bound to hepatocyte surfaces. In this experiment, hepatocytes were prebound with T - L f a t 4 "C in the presence of Ca2+ and Mg", then washed with or without Ca'+, Mg", EGTA, or dextran sulfate (Table 11). As expected, cells retained bound '"I-Lf maximally when washed in buffer containing Ca2+ and Mg2+. In the absence of Ca2+, cells retained half of their surface-bound '"I-Lf whether or not EGTA or Mg" was present. Presumably, 9 - L f lost from these cells had been bound to Ca"-dependent high affinity sites. Dextran sulfate-treated hepatocytes lost > 90% of surface-bound '"I-Lf but only when Ca2+ was absent. When Ca2+ was present, dextran sulfate removed only a portion of the surface-bound radioactivity. Upon titration of dextran sulfate for removal of surface-bound "'I-Lf (Fig. 9), we found that hepatocytes lost > 90% of surface-bound Lf a t dextran sulfate concentrations > 0.3 mg/ml. On the other hand, cells retained 2 50% of their originally bound '"I-Lf when Ca2+ was present regardless of the dextran sulfate concentration tested. Importantly, cells lost almost all of their surface-bound Lf when washed with EGTA and dextran sulfate in combi- nation.

The above data suggested that the Lf bound to cells in two populations, one Ca2+-dependent but insensitive to dextran

TABLE I1 Removal of surface-bound lnctoferrin from hepatocytes

Hepatocytes were incubated for 90 min at 4 "C with '"I-Lf as described under "Experimental Procedures." Cells were washed twice for 10 min in HBS with or without 5 mM CaC12, 5 mM MgC12, 5 mM EGTA, or 0.1% dextran sulfate. The samples were then assayed for bound radioactivity. Values presented represent the mean from duplicate samples f standard deviation. Control, %, represents the amount of lzs1-Lf bound to the cells following washing relative to washing in binding buffer alone.

'2sII-Lactoferrin bound HBS wash supplements

Amount Control

fmol/sample %

Ca", Mg'+ 772 + 23 100 No additions 447 + 50 58 Mg- 479 f 58 62 EGTA 349 f 10 45 Ca2+ 690 + 23 89 M P , EGTA 393 f 11 51 Dextran sulfate 71 k 9 9 Mg'+, dextran sulfate 75 f 1 10 Ca'+, dextran sulfate 367 + 51 48 EGTA, dextran sulfate 39 f 8 5


sulfate, the other Ca'+-independent but sensitive to dextran sulfate. If true, then hepatocytes should bind "'I-Lf by their Ca"-dependent sites even in the presence of dextran sulfate. To test for this possibility, '"I-Lf binding was measured as a

1 2 0 0 1 i 1000 ' " " " I 4 0 0

, O O t L 1 0 250 500 750 1000

Dextran Sulfate (pglrnL)

FIG. 9. Loss of cell-bound Lf as a function of dextran sulfate concentration: effect of Ca2+. Hepatocytes (2 X lofi cell/ml) were incubated at 4 "C with '2sII-Lf (20 pg/ml) for 90 min in HBS containing 5 mM CaCl, and 5 mM MgC1,. Cells were then washed twice, 10 min each wash, in HBS containing either 5 mM CaC12 and 5 mM MgCl, (open squares) or 7.5 mM EGTA and 5 mM MgCl, (closed squares) supplemented with the designated amounts of dextran sulfate. Sam- ples (-lo6 cells) were assayed for bound radioactivity as described under "Experimental Procedures." Symbols represent the mean of duplicate samples that differed by 5 10%.

2.4

1 ' 8 t 1.2

I

0.01 0.1 1 .o 10

CaCI, (rnM)

FIG. 10. Titration of CaZ+ for Lf binding to hepatocytes: effect of dextran sulfate in binding mix. Hepatocytes (-10'/0.5 ml) were incubated with "'II-Lf (2.0 pg/ml) at 4 "C for 90 min in HBS containing 5 mM MgC1, and the designated amount of CaCl, either with (closed squares) or without (open squares) 0.1% dextran sulfate. The cells were then washed in the same buffer at 4 "C, then assayed for bound radioactivity and protein as described under "Experimental Procedures." Symbols represent the mean of duplicate samples that differed by 5 10%.

function of Cay+ in the presence and absence of dextran sulfate (Fig. 10). As expected, hepatocytes bound "'I-Lf in the absence of Ca2+ but not when dextran sulfate was present in the binding mix. In the absence of dextran sulfate, cells doubled the amount of '"I-Lf bound when Cay+ exceeded -0.1 mM. Importantly, hepatocytes also bound I2'I-Lf in the presence of dextran sulfate at Ca2+ 2 0.1 mM. Ca'+ stimulated no additional binding a t concentrations > 1 mM whether or not the binding mix contained dextran sulfate. These results support the notion that hepatocytes bear two classes of Lf binding sites that differ in their requirement for Ca2+ and sensitivity to dextran sulfate.

Endocytosis of Lf by Hepatocytes: Inhibition by Hyperos- molality-We determined whether hepatocytes internalized 12"I-apo-Lf and '"I-holo-Lf at 37 "C. As seen in Table 111, cells bound similar amounts of both ligands at 4 "C, 2 97% of which was stripped from cells by EGTA and dextran sulfate. At 37 "C, cells bound 5 5 times more "'I-Lf than at 4 "C; -80% of this radioactivity was intracellular due to its insen- sitivity to EGTA and dextran sulfate. Notably, cells bound roughly equivalent amounts of "'I-Lf on their surfaces regardless of the incubation temperature. Some cell samples were incubated with Lf in hyperosmotic medium (> 500 mmol/kg with sucrose as osmolite). Such conditions block clathrin-dependent receptor-mediated uptake of various ligands (Daukas and Zigmond, 1985; Heuser and Anderson, 1989; Oka et al., 1989) and constitutive receptor recycling (McAbee et al., 1989) by preventing formation and budding of clathrin-coated pits (Heuser and Anderson, 1989). Hyper- osmolality, however, has no affect on fluid-phase pinocytosis in hepatocytes (Oka et al., 1989). Hyperosmotic medium did not alter '"I-holo-Lf or "'I-apo-Lf binding to cells at 4 "C. On the other hand, hyperosmotic media greatly reduced hepatocyte accumulation of Lf at 37 "C. Cells incubated with I T - Lf a t 37 "C in hyperosmotic media bound roughly the same amount of '"I-Lf as cells incubated a t 4 "C, -90% of which was stripped by EGTA/dextran sulfate wash indicating it was on the cell surface. Thus, hyperosmolality blocked uptake but not binding of "'I-Lf by cells. It appears, therefore, that hepatocytes internalized Lf along a clathrin-dependent pathway; very little, if any, Lf was internalized by way of the clathrin-independent fluid-phase pathway in these cells. These data support the possibility that Lf is endocytosed by hepatocytes via a receptor-mediated mechanism.

TABLE 111 Binding and endocytosis of apo- and holo-Lf: effect of hyperosmolality

Cells (2 X 106/ml BME/BSA) were incubated with ligand at 10 pg/ml for 60 min. After incubation with ligand, cells were washed twice for 10 min in a 4-fold excess of cold HBSS. Samples assayed for associated radioactivity contained -2 X lofi cells. After incubation with ligand, cells were washed twice for 10 min in HBS containing 5 mM EGTA and 0.1% dextran sulfate to remove surface-bound Lf. Samples assayed for radioactivity contained -2 X lo6 cells. Internal counts/min reflected radioactivity associated with cells washed with EGTA and dextran sulfate. Surface radioactivity was calculated by the equation (total) - (internal). Uptake, %, was calculated by the equation, (internal) + (total) X 100. Values represent the mean of duplicate samples * standard deviation.

Ligand Temperature Osmolality "'I-Lf bound

Total Internal Surface Uptake

"C rnrnollkg Apo-Lf 4 280

554 37 271

554 Holo-Lf 4 281

562 37 271

559

4,522 f 229 3,592 & 127

16,080 f 1,240 4,636 f 290 4,109 f 199 4,115 f 59

21,718 & 322 5,463 * 770

fmol/sarnple 137 & 5 119 & 20

12,669 & 300 496 & 42

60 f 6 79 t 1

17,352 * 912 484 f 7

4,385 3,473 3,411 4,140 4,049 4,036 4,366 4,979

%I

3 3

79 11 2 2

80 9

23630 Hepatocyte Binding/Endocytosis of Lactoferrin

DISCUSSION

This report constitutes the first examination of the interaction of "'1-Lf with isolated intact hepatocytes. The primary findings of this study are as follows. 1) Isolated rat hepatocytes bind bovine colostrum "'I-Lf specifically and with mod- erately high affinity. Hepatocytes bind Lf regardless of its iron content in a pH-dependent manner. '"1-apo-Lf and 1251- holo-Lf bind the same sites on hepatocytes. 2) Hepatocytes exhibit two sets of Lf binding sites. One set of sites (-lo6/ cell) binds Lf with high affinity (Kd -20 nM), requires Ca2+ for activity, and binds Lf in the presence of dextran sulfate. EGTA releases Lf bound to these sites. The other set of sites (-107/cell) binds Lf with low affinity (Kd -700 nM), is Ca2+- independent but sensitive to dextran sulfate. 3) Hepatocytes ingest bound Lf regardless of its iron content by a clathrin- dependent mechanism.

We observed two important differences in Lf interaction with intact hepatoctyes compared to the binding of human Lf to isolated whole rat liver plasma membranes (DeBanne et al., 1985). First, intact cells bound Lf optimally at neutral to basic pH (Fig. 8), whereas rat liver plasma membranes bound human Lf optimally at pH 5. Second, lactoperoxidase, lysozyme, and protamine sulfate competed with Lf for binding to membranes. We also found that protamine sulfate and lactoperoxidase inhibited Lf binding to intact cells. In contrast, however, lysozyme had no affect on Lf binding to cells, and cytochrome c, a strongly basic protein, did not block Lf interaction with hepatocytes. In addition, intact cells exhibited high affinity Ca"-dependent binding sites. Isolated liver plasma membranes exhibited Ca2+-dependent increase in human Lf binding, but the equilibrium binding constants of this interaction were not reported even though a high affinity binding component is suggested by their data (DeBanne et al., 1985, Fig. 4). The reasons for these discrepancies are unclear. Isolated intact cells were chosen for this study instead of isolated plasma membranes because they better reflect the physiological interaction of Lf with hepatocytes in situ. Whole hepatic plasma membrane preparations would include plasma membranes from nonparenchymal cells (It0 cells, endothelial cells, Kupffer cells) as well as from hepatocytes. It is certainly possible that nonparenchymal cells may bind Lf in a manner different from hepatocytes. In addition, inside-out membranes present in plasma membrane isolates may increase the fraction of Lf bound via nonspecific ionic bonds obscuring a specific Lf binding "signal." In addition, human and bovine Lf may have some species-specific properties that are exhibited in their interaction with cells and cell membranes. No- tably, bovine Lf did not undergo Ca'+-dependent oligomerization (Fig. 4) as does human Lf (Bennett et al., 1981).

At present, our working hypothesis is that high affinity Lf binding sites consist of bona fide hepatic Lf receptors, whereas low affinity Lf binding sites consist of nonreceptor generic sites that may bind Lf via electrostatic interactions. The dissociation constants for high affinity binding of "'I-apo- and '"I-holo-Lf to hepatocytes (-20 nM) are are similar to the binding constants reported for Lf binding to murine peritoneal macrophages (79 nM; Imber et al., 1982) and to phytohemagglutinin-treated T-cells (83 nM; Mazurier et al., 1989). A Lf binding protein (-100 kDa) has been isolated from these T-cells which exhibit -2 million Lf binding sites/ cell; the majority of these sites are intracellular (Mazurier et al., 1989). If high affinity Ca2+-dependent Lf binding sites on hepatocytes each bind one Lf molecule, then this high affinity receptor (-lO"/cell) constitutes a major hepatocyte surface component, as yet unidentified. It is certainly possible that the hepatic and T-cell Lf receptors are similar or identical.

On the other hand, low affinity Lf binding sites do not require Ca2+ for activity but are sensitive to the effects of dextran sulfate (Table 11; Figs. 5C, 9, 10). Given their relatively low affinity and large number, these sites may consist of anionic cell surface molecules that bind cationic Lf by nonspecific electrostatic interactions. Accordingly, positively charged molecules should compete with Lf for binding these sites but not necessarily with the Ca2+-dependent, high affinity sites. As shown in Fig. 7, however, competition binding with basic proteins was an "all or none" phenomenon at a very large excess of competitor. Thus, the nature of these low affinity sites remains to be determined fully. Lf present in blood at normal concentrations (-20 nM) would interact with hepatocytes predominately by the high affinity Ca2+-dependent binding sites, whereas low affinity Ca2+-independent sites would bind Lf only when blood Lf levels increased substan- tially. Importantly, blood Lf concentrations (-20 nM) are very similar to the calculated Kd for the high affinity Lf binding sites. An important issue that remains to be addressed is how these Lf binding sites are distributed on the surfaces of cells, both in isolated cells and cells in intact liver that retain their unique membrane domains.

The mechanism by which dextran sulfate promotes loss of surface-bound Lf is unknown. Dextran sulfate apparently affects only the low affinity Lf binding sites but not the Ca'+- dependent high affinity sites. This was observed both in the differential removal of Lf prebound to cells by dextran sulfate in the presence and absence of Ca2+ (Table 11; Fig. 9) as well as the differential binding of Lf to cells as a function of Ca'+ in the presence and absence of dextran sulfate (Fig. 10). I t is possible that dextran sulfate interacts with Lf due to their charge differences, thereby removing Lf from cells. Fucoidin blocks liver uptake of blood Lf by this type of mechanism (Imber and Pizzo, 1983). If true, then the interaction of dextran sulfate with Lf must not greatly inhibit L f s ability to bind to the Ca2+-dependent, high affinity sites on hepatocytes. Alternatively, dextran sulfate may interact with cells and thereby block Lf binding. By this mechanism, dextran sulfate apparently blocks hyaluronic acid binding to liver endothelial cells (McGary et al., 1989; Raja et al., 1988) and mannosylated albumin to hepatic mannosyl and scavenger receptors (Jansen et al., 1991). Consistent with the latter possibility, we find that preincubation of hepatocytes with dextran sulfate reduces Lf binding in subsequent binding assays.'

An important finding in this study is that hepatocytes bind and endocytose Lf regardless of its iron content (Table 111). In this way, Lf differs from transferrin, which interacts with cells only in the diferric form (Klausner et al., 1983). This suggests that Lf released into the circulation (apo-Lf -70% of total) by granulocytes may be cleared from the circulation by hepatocytes as readily as holo-Lf. In this way, the liver may modulate blood Lf s functions that require it to bind free iron (e.g. chelation of iron released from reticuloendothelial cells) by removing apo-Lf from the circulation. By this sce- nario, hepatocytes take up apo- and holo-Lf from the circulation not to supplement their nutritional iron requirements but rather to regulate blood Lf s functions as an iron-chelating protein.

Our results corroborate other findings that "'I-holo-Lf injected into the circulation of mice or rats accumulated predominately in hepatocytes compared to nonparenchymal cells (Imber and Pizzo, 1983; Regoeczi et al., 1985), but they are contrary to the results of Courtoy and co-workers (1984) who found that Lf injected into mice was detected microscop-

Unpublished observations.


ically in nonparenchymal cells but not hepatocytes. The basis for this discrepancy is not understood but may be due to differences in types and amounts of Lf used and the methods of measurement. We also found that hepatocyte uptake of Lf required an intact and functional clathrin-based pathway, consistent with the possibility that Lf uptake is receptor- mediated. It also appears that cells internalize Lf largely by the high affinity Ca”-dependent sites rather than the low affinity sites. When cells are prebound with I”1-holo-Lf at 4 “C, then washed in EGTA-containing medium to remove selectively Lf from the high affinity Ca2+-dependent sites, we find that only -15% of ’”I-holo-Lf remaining on the cells (bound to the low affinity sites) is internalized by the cells subsequently at 37 “C. On the other hand, if these cells are washed with dextran sulfate to strip Lf from low affinity Ca’+- independent sites only, then -90% of the I”1-holo-Lf remaining with the cells (bound to the high affinity sites) is internalized subsequently.:‘ Studies are presently underway to characterize fully hepatocyte endocytosis of Lf and to identify an hepatocyte Lf receptor.

Acknowledgments-We thank Dr. Paul Weigel for his support and helpful discussions in the early stages of this study. We also thank Julie Reed and Drs. Sunny Boyd, David Hyde, and Joseph O’Tousa for their helpful discussions during the preparation of this manuscript. We thank Dr. Morton Fuchs for the use of his UV spectrophotometer.

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Documents

Binding and Endocytosis of Apo- and Holo-Lactoferrin by ... · urated (holo-Lf) lactoferrin were used. At 4 “Cy cells bound ‘““I-apo-Lf and “”I-holo-Lf with nearly identi-