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Studies on the mechanism of transferrin iron uptake by rat reticulocytes
553
Biochimica et Biophysica Acta, 632 (1980) 553--561
© Elsevier/North-Holland Biomedical Press
BBA 29419 STUDIES ON THE MECHANISM OF T R A N S F E R R I N IRON UPTAKE BY RAT RETICULOCYTES
ZAHUR ZAMAN, MARIE-JEANNE HEYNEN and ROBRECHT L. VERWILGHEN Department of Medical Research, Catholic University of Leuven, B-3000 Leuven (Belgium)
(Received April 2nd, 1980) Key words: Transferrin; Iron uptake; Endocytosis; (Rat reticulocyte)
Summary Mechanism of transferrin iron uptake by rat reticulocytes was studied using SaFe- and 12SI-labelled rat transferrin. Whereas more than 80% of the reticulocyte-bound SgFe was located in the cytoplasmic fraction, only 25--30% of ~2sIlabelled transferrin was found inside the cells. As shown by the presence of acetylcholine esterase, 10--15% of the cytoplasmic 12sI-labelled transferrin might have been derived from the contamination of this fraction by the plasma membrane fragments. Electron microscopic autoradiography indicated 26% of the cell-bound ~2SI-labelled transferrin to be inside the reticulocytes. Both the electron miscroscopic and biochemical studies showed that the rat reticulocytes endocytosed their plasma membrane independently of transferrin. Sepharoselinked transferrin was found to be capable of delivering S9Fe to the reticulocytes. Our results suggest that penetration of the cell membrane by transferrin is not necessary for the delivery of iron and that, although it might make a contribution to the cellular iron uptake, internalization of transferrin reflects endocytotic activity of the reticulocyte cell membrane.
Iron for the haem biosynthesis, in immature erythroid cells including reticulocytes, is delivered by a plasma iron-carrying protein, transferrin. It is now accepted that the first stage in this delivery o f iron, involves binding of transferrin to specific transferrin receptors on the cell membrane. The mechanism whereby iron is subsequently transferred to the interior of these cells is not y e t fully understood. Earlier, in vitro, studies using 125I-labelled transferrin showed that the radioactive transferrin saturating its binding sites on reticulocytes was fully exchangable with nonradioactive transferrin [1]. It was, therefore, proposed that after binding to its receptors on the cell membrane, transferrin delivers its iron, by an as yet u n k n o w n mechanism, and apotransferrin is liber-
554 ated to repeat the cycle. Biochemical evidence has been presented to support this hypothesis [2--4]. However, morphological studies of Morgan and co-workers [ 5,6], have led them to suggest that the donation of transferrin iron to erythroid cells entails physical internalization of the transferrin-receptor complex by these cells by a process of endocytosis, removal of iron from the endocytotic vesicle and eventual release of apotransferrin into the plasma. In the present study both the biochemical and electron microscopic techniques were used to examine the mechanism of transferrin iron delivery to rat reticulocytes and to determine whether internalization of transferrin was a prerequisite for the transfer of iron to the erythroid cells. We have found that although some transferrin was internalized, the endocytotic activity of the rat reticulocytes could occur independently of transferrin. This suggested that internalization of transferrin might represent a general p h e n o m e n o n of plasma membrane remodelling which is known to occur in maturing reticulocytes [ 7]. Material and Methods
Materials 12sI was obtained from the Radiochemical Centre, Amersham, U.K. SgFe was bought from New England Nuclear Chemicals, Dreieich, F.R.G. Minimum essential medium with Hanks' salts was from Flow Laboratories, Irvine, U.K. Glucose oxidase, 5,5-dithiobis-(2-nitrobenzoic acid), acetylcholine chloride and Triton X-100 were purchased from Sigma Chemical Co. (London), Poole, U.K. Lactoperoxidase was from Worthington Biochemical Corporation, N . J . U . S . A . CM- mad DEAE-Sephadex were obtained from Pharmacia Fine Chemicals, Sweden. Epon 812 resin kit was purchased from Taab, Reading, U.K. Formvar powder 15/95 grade was bought from Ladd, Burlington, VT, U.S.A. All other chemicals were of the highest quality and were bought either from E. Merck, Darmstadt, F.R.G. or J.T. Baker, Deventer, The Netherlands. Methods Rat reticulocytes. Reticulocytosis was induced in male Wistar rats, by three intraperitoneal injection's of neutralized phenylhydrazine (40 mg/kg b o d y weight) during 2 days. Blood, containing 70--75% reticulocytes, was collected over heparin 17 h after the last injection and centrifuged at 1000 × g for 5 min to remove the plasma and b u f f y coat. The sedimented blood cells were washed six times with minimum essential medium containing Hanks' salts (Hanks' medium). Purification o f rat transferrin. Rat transferrin was purified by a combination of previously published methods. This involved precipitation of transferrin b y (NH4)2SO4 between 35--50% saturations [8] followed by chromatographic purification on CM- and DEAE-Sephadex G-50 columns [9]. A46s : A280 ratio of the purified diferric transferrin was 0.0458. This value is characteristic of pure diferric rat transferrin [ 10]. Apotransferrin was prepared by dialysing transferrin solution against 200 mM sodium acetate buffer containing 25 mM EDTA, final pH 5.0. Iodination o f transferrin and retieulocytes. Iodination was carried o u t by two methods. In one of these transferrin and reticulocytes were iodinated
555 according to the procedure of Hubbard and Cohn [11]. In the second method, immobilized glucose oxidase and lactoperoxidase (Enzymobeads from Bio-Rad Laboratories) were used and the reaction mixture contained phosphate buffered saline; 125 pCi Na'2SI in 1.25 pM NaI; 50 tzl Enzymobeads; 10 mM glucose and 3.5 mg transferrin in a total volume of 500 pl. Incubations, carried out at 37°C for 30 min, were terminated by centrifugation to sediment the Enzymobeads. The free '2sI from the s u p e m a t a n t was removed by repeated passage through Sephadex G-25 column (0.9 X 30 cm). 97% of the radioactivity associated with transferrin thus obtained was trichloroacetic acid precipitable. SgFe-labelled transferrin SgFe-labelled transferrin was prepared by incubating a solution of apotransferrin in 250 mM Tris-HC1/5 pM NaHCO3 buffer (final pH 8.0), with a freshly neutralised solution of SgFe-nitrolotriacetic acid complex [12] for I h at 37°C in a total volume of 1 ml. Concentration of Fe was calculated to give 75% saturation of transferrin. Free SgFe was removed by passage through Sephadex G-25 columns, as above. Specific activity of 59Fe was 0.21 gCi/pg Fe and 1 mg transferrin contained 511 • 103 cpm of SgFe. Sepharose-bound transferrin was prepared according to [ 13]. Incubations. 50% (v/v) suspension of the washed reticulocytes in Hanks' medium and one-fifth its volume of '2sI, SgFe-labelled transferrin or '2sIlabelled transferrin were mixed to give a final concentration of 1.4 mg transfertin per ml. These conditions prevent nonspecific binding of transferrin to the reticulocytes [14,15]. From an incubation mixture of 2 ml, 250 pl samples were removed at specified periods and added to 7 ml pre-cooled Hanks' medium. The cells were sedimented by centrifugation at 1000 X g for 5 rain and washed six times with the same medium. Ghosts and cytoplasmic fractions of the haemolysed cells were prepared according to Hanahan and Ekholm [16]. Total uptake of S9Fe and ,2 s I by reticulocytes was estimated by counting known values in a double channel ~/-counter (Berthold, BF 5300). In the case of double-labelled transferrin, the instrument was programmed to make corrections for spill-over of SgFe into the '2sI channel. The spill-over factor was determined according to the manufacturer's instructions. Acetylcholine esterase was determined according to Hanahan and Ekholm [16]. Proteins were determined by the m e t h o d of Lowry et al. [17]. Electron microscopic procedures. Cells were washed six times with Hanks' medium and fixed in 3% glutaraldehyde, in 0.07 M cacodolate buffer/3.7% sucrose (final pH 7.3) for 2 h on ice. The cells were washed in cacodolate buffer, postfixed in 2% osmium tetroxide for 1 h on ice, dehydrated in graded ethanol solutions and embedded in Epon. Sections were collected on slides coated with formvar (0.5% in chloroform). The slides were dipped in an aqueous solution of L-4 (Ilford) to produce a monolayer of the emulsion. The slides were exposed for 30 days and the sections were examined in a Zeiss EM 10 electron microscope w i t h o u t prior staining. The silver grains were counted according to Morgan and Appleton [18]. Results
Iodination of transferrin and reticulocyte plasma membrane. Transferrin and the reticulocyte plasma membrane were iodinated by means of soluble and
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Fig. 1. (a) 1 2 5 I - l a b e l l e d t r a n s f e r r i n u p t a k e b y t h e rat r e t i c u l o c y t c s . A 5 0 % s u s p e n s i o n o f rat r e t i c u l o c y t e s containing 2.8 mg 125I, SgFe-labelled transferrin (20-106 cpm 125I, and 511. 103 cpm S9 Fepermg p r o t e i n ) i n a final v o l u m e o f 2 m l w e r e i n c u b a t e d in a g y r a t o r y w a t e r b a t h at 3 7 ° C . A t s p e c i f i e d t i m e s 2 5 0 p l s a m p l e s w e r e r e m o v e d a n d a d d e d t o 7 m l p r e - c o o l e d H a n k s ' m e d i u m . T h e cells w e r e s e d i m e n t c d b y c e n t r i f u g a t i o n a t 1 0 0 0 X g f o r ~ rain, w a s h e d a n d t h e n h a e m o l y s e d . 1251 and 59 Fe in the total haemoly s a t e , t h e c y t o p l a s m i c f r a c t i o n a n d t h e m e m b r a n e f r a c t i o n w e r e d e t e r m i n e d in a T - c o u n t e r . T h e r e s u l t s w e r e c o n v e r t e d t o c p m / m l c e l l s . T h e m e a n + S . D . is s h o w n b y v e r t i c a l b a r s (n = 1 0 ) . © . . . . . '.~, t o t a l haemolysate; ~----~, membrane fraction; • ...... $, c y t o p l a s m i c fraction. (b) Uptake of transferrinbound 5 9 F e b y rat r e t i c u l o c y t e s . All the conditions are t h e s a m e a s t h o s e d e s c r i b e d f o r Fig. l a . o ..... o, t o t a l h a e m o l y s a t e ; * - - - - - - * membrane fraction; • ...... •, cytoplasmic fraction. For the l a t t e r S . D . w a s t o o s m a l l t o b e s h o w n (n = 1 0 ) .
i m m o b i l i z e d glucose oxidase and lactoperoxidase. B o t h m e t h o d s were effective in iodinating transferrin. 1 7 . 5 - - 2 5 . 1 % o f the added ~2sI was incorporated into the protein. On the other hand, whereas 15% 12sI was incorporated in the reti c u l o c y t e s by the soluble e n z y m e s , i m m o b i l i z e d e n z y m e s effected the incorp o r a t i o n o f o n l y 2.3% o f the added ~2sI. Values o f 0 . 1 4 - - 0 . 3 % were obtained from c o n t r o l e x p e r i m e n t s in w h i c h glucose oxidase, lactoperoxidase or b o t h were omitted. ~2sI and SgFe uptake. Since more than 97% o f 12sI associated with transferrin was c o v a l e n t l y b o u n d to it, the d e t e c t i o n of this radioactivity in a particular
557 fraction represents the presence of transferrin in t h a t fraction. On the other hand, as SgFe was only reversibly bound to transferrin, its localization in a given fraction means that it has originated from transferrin. Transferrin double-labelled with 125I and SgFe was incubated with the rat reticulocytes and the distributions of 125I and SgFe in different cellular fractions were followed as a function of time. The results are shown in Figs. l a and lb. A very rapid binding of transferrin, measured as ~2sI, was observed during the first 10 min of incubation at 37°C. Thereafter, the level of reticulocyte-bound transferrin remained virtually constant, suggesting that the transferrin receptor sites had been saturated. Of the reticulocyte-bound ~2sI, 70--75% was associated with the stromal fraction. The distribution of SgFe presented a completely different picture (Fig. lb). There was almost a linear increase in SgFe uptake by the reticulocytes during the whole of the incubation period. More than 80% of the total reticulocyte SgFe radioactivity could be accounted for in the haemolysate supernatant fraction. However, the level of SgFe in the ghosts remained constant at 15--20% of that present in the total reticulocytes. Acetylcholine esterase activity. It has been found that during the h y p o t o n i c lysis of reticulocytes, fragmentation of plasma membrane may occur [16] and it is possible that some of these fragments may be too small to sediment at 20 000 × g. Therefore acetylcholine esterase, which is a well known membrane marker, was assayed in the total haemolysates and the haemolysate supernat a n t fraction. 10--15% of the total acetylcholine esterase activity found in the reticulocytes was consistently present in the haemolysate supernatant fractions. 59Fe uptake from Sepharose-bound SgFe-labelled transferrin. Sepharosebound transferrin labelled with SgFe and ~2sI was incubated with the rat reticulocytes. The rates of SgFe uptake were compared with those obtained from parallel experiments in which freely soluble SgFe-labelled transferrin was used. Fig. 2 shows that SgFe was taken up from Sepharose-bound transferrin by the cells at a very slow rate. After 1 h only 8--12% of the control was found in the cytoplasm. At the end of the experiment all ~25I could be accounted for in the Sepharose-bound transferrin. Electron microscopic autoradiography. In parallel with the double-labelled transferrin experiment, 12SI-labelled transferrin was incubated with the rat reticulocytes for 30 min and then the cells were processed for electron microscopic autoradiography. The distribution of silver grains was determined according to Morgan and Appleton [18]. Of the grains generated from ~25Ilabelled transferrin 624 were counted. 74% of these were either located on or originated from the cell membrane. In another set of parallel experiments the reticulocytes were incubated, without transferrin, in the presence of ~2sI, soluble glucose oxidase and lactoperoxidase and other reagents for iodination (see Methods). It was anticipated that in this experiment only the plasma membrane of the cells would be labelled with ~25I. However, grain distribution showed that of the 616 grains counted, 21% resided within the cells and 79% originated from the cell membranes. This suggested that fragments of the plasma membrane are internalized independently of transferrin. Internalization of reticulocyte plasma membrane. In order to further sub-
558 T I
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min Fig. 2. 59 Fe u p t a k e b y r a t r e t i c u l o c y t e s f r o m free a n d S e p h a x o s e - b o u n d 59 Fe-labelled t r a n s f e r r i n . A 5 0 % s u s p e n s i o n o f r a t r e t i c u l o c y t e s c o n t a i n i n g 2.8 m g o f S e p h a x o s e - b o u n d 1 2 S i , S 9 F e . l a b e l l e d t r a n s f e r r i n ( 5 1 1 • 103 c p m o f S 9 F e , a n d 20 • 106 c p m o f 1 2 S I / m g t r a n s f e r r i n ) o r f r e e l y s o l u b l e S g F e - l a b e l l e d t r a n s f e r r i n , in a t o t a l v o l u m e o f 2 m l , w e r e i n c u b a t e d at 3 7 ° C . At k n o w n i n t e r v a l s , 2 5 0 #1 s a m p l e s w e r e d i l u t e d w i t h 7 m l H a n k s ' m e d i u m a n d f i l t e r e d t h r o u g h 15 pM m e s h to r e m o v e t h e S e p h a z o s e b e a d s . T h e cells were washed, haemolysed and then counted, o o, f r e e l y s o l u b l e S 9 F e - l a b e l l e d t r a n s f e r r i n ~ • e, S e p h a x o s e - b o u n d 125 I, 59 F e - l a b e l l e d t r a n s f e r r i n . M e a n + S.D. are i n d i c a t e d (n = 10).
stantiate the electron microscopic studies, the rat reticulocytes were labelled with 12sI for 30 min and then reincubated to monitor internalization of their plasma membrane as a function of time. The results (Table I) showed that most of the internalization (18%) of the membrane-bound '2sI occurred during the initial TABLE I INTERNALIZATION
BY RAT RETICULOCYTES
OF THEIR PLASMA MEMBRANE
1 3 0 - - 2 0 0 • 105 r a t r e t i c u l o c y t e s in 1 m l p h o s p h a t e - b u f f e r e d saline w e r e i n c u b a t e d w i t h 3.6 m u n i t s g l u c o s e o x i d a s e ; 3.6 m u n i t s l a c t o p e r o x i d a s e ; 1 0 m M g l u c o s e a n d 10 # C i N a 1 2 5 i , in 2.5 #M N a I . L a c t o p e r o x i d a s e , g l u c o s e o x i d a s e o r b o t h w e r e o m i t t e d f r o m t h e c o n t r o l e x p e r i m e n t s . T h e r e a c t i o n , c a r r i e d o u t in a g y r o t o r y w a t e r b a t h a t 3 7 ° C f o r 30 r a i n , w a s t e r m i n a t e d b y a d d i n g 10 m l p r e c o o l e d H a n k s ' m e d i u m . A f t e r six w a s h e s w i t h t h e s a m e m e d i u m , t h e cells w e r e r e i n c u b a t e d in 1 m l H a n k s ' m e d i u m at 37°C. 2 5 0 /11 s a m p l e s w e r e r e m o v e d a t t h e t i m e s s h o w n . A f t e r six f u r t h e r w a s h e s , t h e cells w e r e l y s e d a n d 12 S I w a s d e t e r m i n e d in t h e m e m b r a n e a n d c y t o p l a s m i c f r a c t i o n s . T h e r e s u l t s axe m e a n s o f t h r e e d u p l i c a t e e x p e r i m e n t s a n d are c o n v e r t e d t o 1 ml e q u i v a l e n t o f cells. Reincubation time (min)
0 15 30 60
1 2 51 epm
• 10 -6
% o f t o t a l 1251 in c y t o p l a s m
Cytoplasm
Membranc
0.395 0.453 0.481 0.520
1.780 1.720 1.694 1.655
18.20 20.84 22.11 23.91
559 iodination period. Reincubation of the labelled reticulocytes led to an increase in internalization of l~sI to a b o u t 24%. That the radioactivity in the reticulocytes did not originate from free ~2sI was shown by the control experiments in which only 42000-+ 4500 cpm were found to be associated with the cells. Therefore, these results confirmed that the reticulocytes internalize their plasma membrane even in the absence of transferrin. Discussion Currently two mechanisms, for the delivery of transferrin-bound iron to reticulocytes, are being considered. One of these requires a specific acceptordependent binding of transferrin to the reticulocytes followed by a plasma membrane-mediated release and transfer of the iron to the interior of the cell. Studies supporting this mechanism have shown that the receptor-bound transferrin is freely exchangable with the transferrin in the medium [ 1 ] and that the reticulocyte plasma membrane is capable of causing a release of iron from transferrin [2,3]. The second mechanism suggests that the transferrin-receptor complex is internalized by endocytosis or a microtubular system [19]. This is followed by transfer of iron to the cytoplasm and release of the apotransferrin molecule into the plasma. Evidence supporting this hypothesis is obtained mainly from morphological studies [5,18]. At face value the results from the present study may be taken to support both mechanisms. However, we, like Loh et al. [4], have found that the movements of S9Fe and transferrin into the cell are not coordinated. While cytoplasmic S9Fe closely follows the course of S9Fe radioactivity associated with whole cells at all times (Fig. la), only 25--30% of the reticulocyte-bound transferrin is in the cytoplasm (Fig. lb). When this value of transferrin found in haemolysate supernatant is corrected for molecules of transferrin which might be associated with the fragments of plasma membrane, as revealed by the presence of acetylcholine esterase, it is reduced to 10--20%. Electron microscopic autoradiography of the cell sections obtained from the reticulocytes incubated with 12sIlabelled transferrin for 30 min and from those which had been iodinated for the same period showed that 26 and 21% grains, respectively, were located inside the cells and in both cases grains were superimposed on vesicles. Since direct iodination of the reticulocytes was expected to label only the exterior of the cell membranes, the presence of the radioactive grains inside the cells suggested that the reticulocyte membrane is e n d o c y t o s e d independently of transferrin. This conclusion was further supported by the results of experiments in which externally-located and covalently-bound radioactive label on the rat reticulocyte plasma membrane was found to be internalized in the absence of transferrin (Table I). Using an entirely different approach Sullivan et al. [20] have also shown that the endocytotic activity o f the rat reticulocyte is indepedent of transferrin. We suggest that a significant amount of transferrin internalization represents a general p h e n o m e n o n of reticulocyte membrane remodelling, rather than a prerequisite for the delivery of iron to the reticulocyte. This idea is supported n o t only by our results discussed above b u t also by recent reports in which it has been shown that maturing reticulocytes are very active in endocytotic and
560 exocytotic activities [7,21]. Since only those substances which bind to the reticulocyte membrane are internalized, it suggests that any substance capable of binding to the reticulocyte cell membrane might be endocytosed. Thus, endocytosis of gold particles [7] and concanavalin A [21] might be fortuitous events secondary to membrane remodelling or they might reflect an active response by the cell to eliminate these substances. Similarly, in the case of transferrin its internalization might be brought about as a consequence of gradual transferrin-receptor elimination which occurs during reticulocyte maturation, and it is also possible that molecules of transferrin altered by chemical modification or total or partial denaturation are preferentially internalized. As supportive evidence for this possibility, Milsom and Batey [22] have found that only denatured transferrin is taken up by the rat liver. The inability of immobilized transferrin to donate iron to rabbit reticulocytes has been interpreted to mean that internalization of transferrin is obligatory for the delivery of iron [5]. However, Lob et al. [4] and Glass et al. [23] have shown that a significant a m o u n t of iron from the Sepharose-bound transferrin was taken up by the rabbit and murine reticulocytes. Using rat reticulocytes we have found that the Sepharose-bound rat transferrin was capable of delivering iron albeit only 8--12% of that delivered by free transferrin (Fig. 2). These results show that internalization of transferrin is not absolutely necessary for the delivery of iron to the rat reticulocytes. On the other hand negative results would not necessarily have proven the opposite. One of the reasons [23] for this is that the bulkiness of the ligand to which transferrin is bound might prevent transferrin-receptor complex formation on the cell membrane. This is supported by our iodination experiments in which immobilized lactoperoxidase and glucose oxidase failed to iodinate the rat reticulocyte membrane to a significant extent. In conclusion, while confirming internalization of transferrin by the rat reticulocytes, our data suggest that it might be a secondary consequence of transferrin-independent endocytotic activity of the maturing reticulocyte cell membrane. Although it would contribute to the cellular iron uptake, internalization of transferrin is unlikely to represent the sole mechanism for the delivery of iron. The ability of immobilized-transferrin to transport iron and the lack of coordination, between the movements of iron and transferrin into the cell, support the membrane-mediated iron transfer mechanism [1] in which physical entry of transferrin into the reticulocytes is not an absolute requirement. Since it cannot be ruled out that both mechanisms operate in vivo, it would be interesting to know the extent of the contribution made by each mechanism. Acknowledgements We thank the Nationaal Fonds voor Geneeskundig Wetenschappelijk Onderzoek for a research grant (2.002,76) and V. Van Duppen for technical assistance. References 1 J a n d l , J . H . a n d K a t z , J . H . ( 1 9 6 3 ) J. Clin. Invest. 42, 3 1 4 - - 3 2 6 2 E g y c d , A. ( 1 9 7 5 ) A c t a Biochira. B i o p h y s . A c a d . Sci. H u n g . 9, 4 3 - - 5 2
561
3 W o r k m a n , E . F . a n d B a t e s , G.W. ( 1 9 7 5 ) in P r o t e i n s o f I r o n S t o r a g e a n d T r a n s p o r t in B i o c h e m i s t r y a n d Medicine (R.R, Crichton, ed.), pp. 155--160, North Holland Publishing Co., Amsterdam 4 L o h , T . T . , Y e u n g , Y . G . a n d Y e u n g , D. ( 1 9 7 7 ) B i o e h i m . B i o p h y s . A c t a 4 7 1 , 1 1 8 - - 1 2 4 5 H e m m a p l a r d h , D. a n d M o r g a n , E . H . ( 1 9 7 7 ) Br. J. H a e m a t o l . 3 6 , 8 5 - - 9 6 6 V a n B o c k x m e e r , F.M. a n d M o r g a n , E . H . ( 1 9 7 9 ) B i o c h i m . B i o p h y s . A c t a 5 8 4 , 7 6 - - 8 3 7 G a s k o , O. a n d D a n o n , D. ( 1 9 7 4 ) Br. J. H a e m a t o l . 2 8 , 4 6 3 - - 4 7 0 8 M o r g a n , E . H . ( 1 9 6 4 ) J. P h y s i o l . 1 7 1 , 2 6 - - 4 1 9 H a t t o n , M.W.C., R e g e o c z i , E., W o n g , K . - L . a n d K r a a y , G . J . ( 1 9 7 7 ) B i o c h c m . G e n e t . 15, 6 2 1 - - - 6 4 0 10 H u e b e r s , H., H u e b e r s , E., Csiba, E. a n d F i n c h , C.A. ( 1 9 7 8 ) J. Clin. I n v e s t . 6 2 , 9 4 4 - - 9 5 1 11 H u b b a r d , A . L . a n d C o h n , Z . A . ( 1 9 7 2 ) J. Cell. Biol. 5 5 , 3 9 0 - - 4 0 5 12 H a r r i s , D.C. a n d Aisen, P. ( 1 9 7 5 ) B i o c h e m i s t r y 14, 2 6 2 - - 2 6 8 13 C u a t r a c a s a s , P. ( 1 9 7 0 ) J. Biol. C h e m . 2 4 5 , 3 0 5 9 - - 3 0 6 5 14 S p e y e r , B.E. a n d F i e l d i n g . J. ( 1 9 7 4 ) Bioct~im. B i o p h y s . A c t a 3 3 2 , 1 9 2 - - 2 0 0 1 5 F i e l d i n g , J. a n d S p e y e r , B.E. ( 1 9 7 4 ) B i o c h i m . B i o p h y s . A c t a 3 3 6 , 3 8 7 - - 3 9 6 1 6 H a n a h a n , D.J. a n d E k h o l m , J . E . ( 1 9 7 4 ) M e t h o d s E n z y m o l . 31, 1 6 8 - - 1 8 0 17 L o w r y , O . H . , R o s e b r o u g h , N . J . , F a r r , A . L . a n d R a n d a l l , R . J . ( 1 9 5 1 ) J. Biol. C h e m . 1 9 3 , 2 6 5 - - 2 7 5 18 M o r g a n , E . H . a n d A p p l e t o n , T.C. ( 1 9 6 9 ) N a t u r e 2 2 3 , 1 3 7 1 - - 1 3 7 2 19 H e m m a p l a r d h , D., Kailis, S.G. a n d M o r g a n , E.M. ( 1 9 7 4 ) Br. J. H a e m a t o l . 2 8 , 5 3 - - 6 5 20 Sullivan, A.L., Grasso, J.A. and Weintraub, L.R. (1976) Blood 47, 133--143 21 Z w e i g , S. a n d S i n g e r . S.J. ( 1 9 7 9 ) J. Cell Biol. 8 0 , 4 8 7 - - 4 9 1 2 2 M i l s o m , J . P . a n d B a t e y , R . G . ( 1 9 7 9 ) B i o c h e m . J. 1 8 2 , 1 1 7 - - 1 2 5 2 3 Glass, J., N u n e z , M . T . a n d R o b i s o n , S.H. ( 1 9 7 7 ) B i o c h e m . B i o p h y s . Res. C o m m u n . 7 5 , 2 2 6 - - 2 3 2
Biochimica et Biophysica Acta, 632 (1980) 553--561
© Elsevier/North-Holland Biomedical Press
BBA 29419 STUDIES ON THE MECHANISM OF T R A N S F E R R I N IRON UPTAKE BY RAT RETICULOCYTES
ZAHUR ZAMAN, MARIE-JEANNE HEYNEN and ROBRECHT L. VERWILGHEN Department of Medical Research, Catholic University of Leuven, B-3000 Leuven (Belgium)
(Received April 2nd, 1980) Key words: Transferrin; Iron uptake; Endocytosis; (Rat reticulocyte)
Summary Mechanism of transferrin iron uptake by rat reticulocytes was studied using SaFe- and 12SI-labelled rat transferrin. Whereas more than 80% of the reticulocyte-bound SgFe was located in the cytoplasmic fraction, only 25--30% of ~2sIlabelled transferrin was found inside the cells. As shown by the presence of acetylcholine esterase, 10--15% of the cytoplasmic 12sI-labelled transferrin might have been derived from the contamination of this fraction by the plasma membrane fragments. Electron microscopic autoradiography indicated 26% of the cell-bound ~2SI-labelled transferrin to be inside the reticulocytes. Both the electron miscroscopic and biochemical studies showed that the rat reticulocytes endocytosed their plasma membrane independently of transferrin. Sepharoselinked transferrin was found to be capable of delivering S9Fe to the reticulocytes. Our results suggest that penetration of the cell membrane by transferrin is not necessary for the delivery of iron and that, although it might make a contribution to the cellular iron uptake, internalization of transferrin reflects endocytotic activity of the reticulocyte cell membrane.
Iron for the haem biosynthesis, in immature erythroid cells including reticulocytes, is delivered by a plasma iron-carrying protein, transferrin. It is now accepted that the first stage in this delivery o f iron, involves binding of transferrin to specific transferrin receptors on the cell membrane. The mechanism whereby iron is subsequently transferred to the interior of these cells is not y e t fully understood. Earlier, in vitro, studies using 125I-labelled transferrin showed that the radioactive transferrin saturating its binding sites on reticulocytes was fully exchangable with nonradioactive transferrin [1]. It was, therefore, proposed that after binding to its receptors on the cell membrane, transferrin delivers its iron, by an as yet u n k n o w n mechanism, and apotransferrin is liber-
554 ated to repeat the cycle. Biochemical evidence has been presented to support this hypothesis [2--4]. However, morphological studies of Morgan and co-workers [ 5,6], have led them to suggest that the donation of transferrin iron to erythroid cells entails physical internalization of the transferrin-receptor complex by these cells by a process of endocytosis, removal of iron from the endocytotic vesicle and eventual release of apotransferrin into the plasma. In the present study both the biochemical and electron microscopic techniques were used to examine the mechanism of transferrin iron delivery to rat reticulocytes and to determine whether internalization of transferrin was a prerequisite for the transfer of iron to the erythroid cells. We have found that although some transferrin was internalized, the endocytotic activity of the rat reticulocytes could occur independently of transferrin. This suggested that internalization of transferrin might represent a general p h e n o m e n o n of plasma membrane remodelling which is known to occur in maturing reticulocytes [ 7]. Material and Methods
Materials 12sI was obtained from the Radiochemical Centre, Amersham, U.K. SgFe was bought from New England Nuclear Chemicals, Dreieich, F.R.G. Minimum essential medium with Hanks' salts was from Flow Laboratories, Irvine, U.K. Glucose oxidase, 5,5-dithiobis-(2-nitrobenzoic acid), acetylcholine chloride and Triton X-100 were purchased from Sigma Chemical Co. (London), Poole, U.K. Lactoperoxidase was from Worthington Biochemical Corporation, N . J . U . S . A . CM- mad DEAE-Sephadex were obtained from Pharmacia Fine Chemicals, Sweden. Epon 812 resin kit was purchased from Taab, Reading, U.K. Formvar powder 15/95 grade was bought from Ladd, Burlington, VT, U.S.A. All other chemicals were of the highest quality and were bought either from E. Merck, Darmstadt, F.R.G. or J.T. Baker, Deventer, The Netherlands. Methods Rat reticulocytes. Reticulocytosis was induced in male Wistar rats, by three intraperitoneal injection's of neutralized phenylhydrazine (40 mg/kg b o d y weight) during 2 days. Blood, containing 70--75% reticulocytes, was collected over heparin 17 h after the last injection and centrifuged at 1000 × g for 5 min to remove the plasma and b u f f y coat. The sedimented blood cells were washed six times with minimum essential medium containing Hanks' salts (Hanks' medium). Purification o f rat transferrin. Rat transferrin was purified by a combination of previously published methods. This involved precipitation of transferrin b y (NH4)2SO4 between 35--50% saturations [8] followed by chromatographic purification on CM- and DEAE-Sephadex G-50 columns [9]. A46s : A280 ratio of the purified diferric transferrin was 0.0458. This value is characteristic of pure diferric rat transferrin [ 10]. Apotransferrin was prepared by dialysing transferrin solution against 200 mM sodium acetate buffer containing 25 mM EDTA, final pH 5.0. Iodination o f transferrin and retieulocytes. Iodination was carried o u t by two methods. In one of these transferrin and reticulocytes were iodinated
555 according to the procedure of Hubbard and Cohn [11]. In the second method, immobilized glucose oxidase and lactoperoxidase (Enzymobeads from Bio-Rad Laboratories) were used and the reaction mixture contained phosphate buffered saline; 125 pCi Na'2SI in 1.25 pM NaI; 50 tzl Enzymobeads; 10 mM glucose and 3.5 mg transferrin in a total volume of 500 pl. Incubations, carried out at 37°C for 30 min, were terminated by centrifugation to sediment the Enzymobeads. The free '2sI from the s u p e m a t a n t was removed by repeated passage through Sephadex G-25 column (0.9 X 30 cm). 97% of the radioactivity associated with transferrin thus obtained was trichloroacetic acid precipitable. SgFe-labelled transferrin SgFe-labelled transferrin was prepared by incubating a solution of apotransferrin in 250 mM Tris-HC1/5 pM NaHCO3 buffer (final pH 8.0), with a freshly neutralised solution of SgFe-nitrolotriacetic acid complex [12] for I h at 37°C in a total volume of 1 ml. Concentration of Fe was calculated to give 75% saturation of transferrin. Free SgFe was removed by passage through Sephadex G-25 columns, as above. Specific activity of 59Fe was 0.21 gCi/pg Fe and 1 mg transferrin contained 511 • 103 cpm of SgFe. Sepharose-bound transferrin was prepared according to [ 13]. Incubations. 50% (v/v) suspension of the washed reticulocytes in Hanks' medium and one-fifth its volume of '2sI, SgFe-labelled transferrin or '2sIlabelled transferrin were mixed to give a final concentration of 1.4 mg transfertin per ml. These conditions prevent nonspecific binding of transferrin to the reticulocytes [14,15]. From an incubation mixture of 2 ml, 250 pl samples were removed at specified periods and added to 7 ml pre-cooled Hanks' medium. The cells were sedimented by centrifugation at 1000 X g for 5 rain and washed six times with the same medium. Ghosts and cytoplasmic fractions of the haemolysed cells were prepared according to Hanahan and Ekholm [16]. Total uptake of S9Fe and ,2 s I by reticulocytes was estimated by counting known values in a double channel ~/-counter (Berthold, BF 5300). In the case of double-labelled transferrin, the instrument was programmed to make corrections for spill-over of SgFe into the '2sI channel. The spill-over factor was determined according to the manufacturer's instructions. Acetylcholine esterase was determined according to Hanahan and Ekholm [16]. Proteins were determined by the m e t h o d of Lowry et al. [17]. Electron microscopic procedures. Cells were washed six times with Hanks' medium and fixed in 3% glutaraldehyde, in 0.07 M cacodolate buffer/3.7% sucrose (final pH 7.3) for 2 h on ice. The cells were washed in cacodolate buffer, postfixed in 2% osmium tetroxide for 1 h on ice, dehydrated in graded ethanol solutions and embedded in Epon. Sections were collected on slides coated with formvar (0.5% in chloroform). The slides were dipped in an aqueous solution of L-4 (Ilford) to produce a monolayer of the emulsion. The slides were exposed for 30 days and the sections were examined in a Zeiss EM 10 electron microscope w i t h o u t prior staining. The silver grains were counted according to Morgan and Appleton [18]. Results
Iodination of transferrin and reticulocyte plasma membrane. Transferrin and the reticulocyte plasma membrane were iodinated by means of soluble and
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Fig. 1. (a) 1 2 5 I - l a b e l l e d t r a n s f e r r i n u p t a k e b y t h e rat r e t i c u l o c y t c s . A 5 0 % s u s p e n s i o n o f rat r e t i c u l o c y t e s containing 2.8 mg 125I, SgFe-labelled transferrin (20-106 cpm 125I, and 511. 103 cpm S9 Fepermg p r o t e i n ) i n a final v o l u m e o f 2 m l w e r e i n c u b a t e d in a g y r a t o r y w a t e r b a t h at 3 7 ° C . A t s p e c i f i e d t i m e s 2 5 0 p l s a m p l e s w e r e r e m o v e d a n d a d d e d t o 7 m l p r e - c o o l e d H a n k s ' m e d i u m . T h e cells w e r e s e d i m e n t c d b y c e n t r i f u g a t i o n a t 1 0 0 0 X g f o r ~ rain, w a s h e d a n d t h e n h a e m o l y s e d . 1251 and 59 Fe in the total haemoly s a t e , t h e c y t o p l a s m i c f r a c t i o n a n d t h e m e m b r a n e f r a c t i o n w e r e d e t e r m i n e d in a T - c o u n t e r . T h e r e s u l t s w e r e c o n v e r t e d t o c p m / m l c e l l s . T h e m e a n + S . D . is s h o w n b y v e r t i c a l b a r s (n = 1 0 ) . © . . . . . '.~, t o t a l haemolysate; ~----~, membrane fraction; • ...... $, c y t o p l a s m i c fraction. (b) Uptake of transferrinbound 5 9 F e b y rat r e t i c u l o c y t e s . All the conditions are t h e s a m e a s t h o s e d e s c r i b e d f o r Fig. l a . o ..... o, t o t a l h a e m o l y s a t e ; * - - - - - - * membrane fraction; • ...... •, cytoplasmic fraction. For the l a t t e r S . D . w a s t o o s m a l l t o b e s h o w n (n = 1 0 ) .
i m m o b i l i z e d glucose oxidase and lactoperoxidase. B o t h m e t h o d s were effective in iodinating transferrin. 1 7 . 5 - - 2 5 . 1 % o f the added ~2sI was incorporated into the protein. On the other hand, whereas 15% 12sI was incorporated in the reti c u l o c y t e s by the soluble e n z y m e s , i m m o b i l i z e d e n z y m e s effected the incorp o r a t i o n o f o n l y 2.3% o f the added ~2sI. Values o f 0 . 1 4 - - 0 . 3 % were obtained from c o n t r o l e x p e r i m e n t s in w h i c h glucose oxidase, lactoperoxidase or b o t h were omitted. ~2sI and SgFe uptake. Since more than 97% o f 12sI associated with transferrin was c o v a l e n t l y b o u n d to it, the d e t e c t i o n of this radioactivity in a particular
557 fraction represents the presence of transferrin in t h a t fraction. On the other hand, as SgFe was only reversibly bound to transferrin, its localization in a given fraction means that it has originated from transferrin. Transferrin double-labelled with 125I and SgFe was incubated with the rat reticulocytes and the distributions of 125I and SgFe in different cellular fractions were followed as a function of time. The results are shown in Figs. l a and lb. A very rapid binding of transferrin, measured as ~2sI, was observed during the first 10 min of incubation at 37°C. Thereafter, the level of reticulocyte-bound transferrin remained virtually constant, suggesting that the transferrin receptor sites had been saturated. Of the reticulocyte-bound ~2sI, 70--75% was associated with the stromal fraction. The distribution of SgFe presented a completely different picture (Fig. lb). There was almost a linear increase in SgFe uptake by the reticulocytes during the whole of the incubation period. More than 80% of the total reticulocyte SgFe radioactivity could be accounted for in the haemolysate supernatant fraction. However, the level of SgFe in the ghosts remained constant at 15--20% of that present in the total reticulocytes. Acetylcholine esterase activity. It has been found that during the h y p o t o n i c lysis of reticulocytes, fragmentation of plasma membrane may occur [16] and it is possible that some of these fragments may be too small to sediment at 20 000 × g. Therefore acetylcholine esterase, which is a well known membrane marker, was assayed in the total haemolysates and the haemolysate supernat a n t fraction. 10--15% of the total acetylcholine esterase activity found in the reticulocytes was consistently present in the haemolysate supernatant fractions. 59Fe uptake from Sepharose-bound SgFe-labelled transferrin. Sepharosebound transferrin labelled with SgFe and ~2sI was incubated with the rat reticulocytes. The rates of SgFe uptake were compared with those obtained from parallel experiments in which freely soluble SgFe-labelled transferrin was used. Fig. 2 shows that SgFe was taken up from Sepharose-bound transferrin by the cells at a very slow rate. After 1 h only 8--12% of the control was found in the cytoplasm. At the end of the experiment all ~25I could be accounted for in the Sepharose-bound transferrin. Electron microscopic autoradiography. In parallel with the double-labelled transferrin experiment, 12SI-labelled transferrin was incubated with the rat reticulocytes for 30 min and then the cells were processed for electron microscopic autoradiography. The distribution of silver grains was determined according to Morgan and Appleton [18]. Of the grains generated from ~25Ilabelled transferrin 624 were counted. 74% of these were either located on or originated from the cell membrane. In another set of parallel experiments the reticulocytes were incubated, without transferrin, in the presence of ~2sI, soluble glucose oxidase and lactoperoxidase and other reagents for iodination (see Methods). It was anticipated that in this experiment only the plasma membrane of the cells would be labelled with ~25I. However, grain distribution showed that of the 616 grains counted, 21% resided within the cells and 79% originated from the cell membranes. This suggested that fragments of the plasma membrane are internalized independently of transferrin. Internalization of reticulocyte plasma membrane. In order to further sub-
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min Fig. 2. 59 Fe u p t a k e b y r a t r e t i c u l o c y t e s f r o m free a n d S e p h a x o s e - b o u n d 59 Fe-labelled t r a n s f e r r i n . A 5 0 % s u s p e n s i o n o f r a t r e t i c u l o c y t e s c o n t a i n i n g 2.8 m g o f S e p h a x o s e - b o u n d 1 2 S i , S 9 F e . l a b e l l e d t r a n s f e r r i n ( 5 1 1 • 103 c p m o f S 9 F e , a n d 20 • 106 c p m o f 1 2 S I / m g t r a n s f e r r i n ) o r f r e e l y s o l u b l e S g F e - l a b e l l e d t r a n s f e r r i n , in a t o t a l v o l u m e o f 2 m l , w e r e i n c u b a t e d at 3 7 ° C . At k n o w n i n t e r v a l s , 2 5 0 #1 s a m p l e s w e r e d i l u t e d w i t h 7 m l H a n k s ' m e d i u m a n d f i l t e r e d t h r o u g h 15 pM m e s h to r e m o v e t h e S e p h a z o s e b e a d s . T h e cells were washed, haemolysed and then counted, o o, f r e e l y s o l u b l e S 9 F e - l a b e l l e d t r a n s f e r r i n ~ • e, S e p h a x o s e - b o u n d 125 I, 59 F e - l a b e l l e d t r a n s f e r r i n . M e a n + S.D. are i n d i c a t e d (n = 10).
stantiate the electron microscopic studies, the rat reticulocytes were labelled with 12sI for 30 min and then reincubated to monitor internalization of their plasma membrane as a function of time. The results (Table I) showed that most of the internalization (18%) of the membrane-bound '2sI occurred during the initial TABLE I INTERNALIZATION
BY RAT RETICULOCYTES
OF THEIR PLASMA MEMBRANE
1 3 0 - - 2 0 0 • 105 r a t r e t i c u l o c y t e s in 1 m l p h o s p h a t e - b u f f e r e d saline w e r e i n c u b a t e d w i t h 3.6 m u n i t s g l u c o s e o x i d a s e ; 3.6 m u n i t s l a c t o p e r o x i d a s e ; 1 0 m M g l u c o s e a n d 10 # C i N a 1 2 5 i , in 2.5 #M N a I . L a c t o p e r o x i d a s e , g l u c o s e o x i d a s e o r b o t h w e r e o m i t t e d f r o m t h e c o n t r o l e x p e r i m e n t s . T h e r e a c t i o n , c a r r i e d o u t in a g y r o t o r y w a t e r b a t h a t 3 7 ° C f o r 30 r a i n , w a s t e r m i n a t e d b y a d d i n g 10 m l p r e c o o l e d H a n k s ' m e d i u m . A f t e r six w a s h e s w i t h t h e s a m e m e d i u m , t h e cells w e r e r e i n c u b a t e d in 1 m l H a n k s ' m e d i u m at 37°C. 2 5 0 /11 s a m p l e s w e r e r e m o v e d a t t h e t i m e s s h o w n . A f t e r six f u r t h e r w a s h e s , t h e cells w e r e l y s e d a n d 12 S I w a s d e t e r m i n e d in t h e m e m b r a n e a n d c y t o p l a s m i c f r a c t i o n s . T h e r e s u l t s axe m e a n s o f t h r e e d u p l i c a t e e x p e r i m e n t s a n d are c o n v e r t e d t o 1 ml e q u i v a l e n t o f cells. Reincubation time (min)
0 15 30 60
1 2 51 epm
• 10 -6
% o f t o t a l 1251 in c y t o p l a s m
Cytoplasm
Membranc
0.395 0.453 0.481 0.520
1.780 1.720 1.694 1.655
18.20 20.84 22.11 23.91
559 iodination period. Reincubation of the labelled reticulocytes led to an increase in internalization of l~sI to a b o u t 24%. That the radioactivity in the reticulocytes did not originate from free ~2sI was shown by the control experiments in which only 42000-+ 4500 cpm were found to be associated with the cells. Therefore, these results confirmed that the reticulocytes internalize their plasma membrane even in the absence of transferrin. Discussion Currently two mechanisms, for the delivery of transferrin-bound iron to reticulocytes, are being considered. One of these requires a specific acceptordependent binding of transferrin to the reticulocytes followed by a plasma membrane-mediated release and transfer of the iron to the interior of the cell. Studies supporting this mechanism have shown that the receptor-bound transferrin is freely exchangable with the transferrin in the medium [ 1 ] and that the reticulocyte plasma membrane is capable of causing a release of iron from transferrin [2,3]. The second mechanism suggests that the transferrin-receptor complex is internalized by endocytosis or a microtubular system [19]. This is followed by transfer of iron to the cytoplasm and release of the apotransferrin molecule into the plasma. Evidence supporting this hypothesis is obtained mainly from morphological studies [5,18]. At face value the results from the present study may be taken to support both mechanisms. However, we, like Loh et al. [4], have found that the movements of S9Fe and transferrin into the cell are not coordinated. While cytoplasmic S9Fe closely follows the course of S9Fe radioactivity associated with whole cells at all times (Fig. la), only 25--30% of the reticulocyte-bound transferrin is in the cytoplasm (Fig. lb). When this value of transferrin found in haemolysate supernatant is corrected for molecules of transferrin which might be associated with the fragments of plasma membrane, as revealed by the presence of acetylcholine esterase, it is reduced to 10--20%. Electron microscopic autoradiography of the cell sections obtained from the reticulocytes incubated with 12sIlabelled transferrin for 30 min and from those which had been iodinated for the same period showed that 26 and 21% grains, respectively, were located inside the cells and in both cases grains were superimposed on vesicles. Since direct iodination of the reticulocytes was expected to label only the exterior of the cell membranes, the presence of the radioactive grains inside the cells suggested that the reticulocyte membrane is e n d o c y t o s e d independently of transferrin. This conclusion was further supported by the results of experiments in which externally-located and covalently-bound radioactive label on the rat reticulocyte plasma membrane was found to be internalized in the absence of transferrin (Table I). Using an entirely different approach Sullivan et al. [20] have also shown that the endocytotic activity o f the rat reticulocyte is indepedent of transferrin. We suggest that a significant amount of transferrin internalization represents a general p h e n o m e n o n of reticulocyte membrane remodelling, rather than a prerequisite for the delivery of iron to the reticulocyte. This idea is supported n o t only by our results discussed above b u t also by recent reports in which it has been shown that maturing reticulocytes are very active in endocytotic and
560 exocytotic activities [7,21]. Since only those substances which bind to the reticulocyte membrane are internalized, it suggests that any substance capable of binding to the reticulocyte cell membrane might be endocytosed. Thus, endocytosis of gold particles [7] and concanavalin A [21] might be fortuitous events secondary to membrane remodelling or they might reflect an active response by the cell to eliminate these substances. Similarly, in the case of transferrin its internalization might be brought about as a consequence of gradual transferrin-receptor elimination which occurs during reticulocyte maturation, and it is also possible that molecules of transferrin altered by chemical modification or total or partial denaturation are preferentially internalized. As supportive evidence for this possibility, Milsom and Batey [22] have found that only denatured transferrin is taken up by the rat liver. The inability of immobilized transferrin to donate iron to rabbit reticulocytes has been interpreted to mean that internalization of transferrin is obligatory for the delivery of iron [5]. However, Lob et al. [4] and Glass et al. [23] have shown that a significant a m o u n t of iron from the Sepharose-bound transferrin was taken up by the rabbit and murine reticulocytes. Using rat reticulocytes we have found that the Sepharose-bound rat transferrin was capable of delivering iron albeit only 8--12% of that delivered by free transferrin (Fig. 2). These results show that internalization of transferrin is not absolutely necessary for the delivery of iron to the rat reticulocytes. On the other hand negative results would not necessarily have proven the opposite. One of the reasons [23] for this is that the bulkiness of the ligand to which transferrin is bound might prevent transferrin-receptor complex formation on the cell membrane. This is supported by our iodination experiments in which immobilized lactoperoxidase and glucose oxidase failed to iodinate the rat reticulocyte membrane to a significant extent. In conclusion, while confirming internalization of transferrin by the rat reticulocytes, our data suggest that it might be a secondary consequence of transferrin-independent endocytotic activity of the maturing reticulocyte cell membrane. Although it would contribute to the cellular iron uptake, internalization of transferrin is unlikely to represent the sole mechanism for the delivery of iron. The ability of immobilized-transferrin to transport iron and the lack of coordination, between the movements of iron and transferrin into the cell, support the membrane-mediated iron transfer mechanism [1] in which physical entry of transferrin into the reticulocytes is not an absolute requirement. Since it cannot be ruled out that both mechanisms operate in vivo, it would be interesting to know the extent of the contribution made by each mechanism. Acknowledgements We thank the Nationaal Fonds voor Geneeskundig Wetenschappelijk Onderzoek for a research grant (2.002,76) and V. Van Duppen for technical assistance. References 1 J a n d l , J . H . a n d K a t z , J . H . ( 1 9 6 3 ) J. Clin. Invest. 42, 3 1 4 - - 3 2 6 2 E g y c d , A. ( 1 9 7 5 ) A c t a Biochira. B i o p h y s . A c a d . Sci. H u n g . 9, 4 3 - - 5 2
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