Clinical Science and Molecular Medicine (1 977) 52, 87-96.

Binding of transferrin and uptake of iron by rat erythroid cells in vitro N . J. VERHOEF A N D P. J. N O O R D E L O O S Department of Chemical Pathology. Faculty of Medicine, Erasmus University Rotterdam, Rotterdam, The Netherlands (Received 3 May 1976; accepted 24 August 1976)

subcellular distribution of the radioactivities could be observed. 6. It wasconcludedthat the membranefraction contains appreciable amounts of 59Fe not bound to Iz Wabelled transferrin, which indicates that dissociation of the iron-transferrin complex is one of the earlier steps in the mechanism of iron uptake by erythroid cells.

summary 1. The binding of transferrin and the uptakeof iron by rat bone-marrow-cell suspensions was investigated by the use of transferrin doubly labelled with lZsIand 59Fe. 2. The pattern- of transferrin binding was found to depend on the transferrin concentration in the incubation medium. At relatively low concentrations, binding of transferrin at 04'C was lower than the binding at 37°C. At higher concentrations no difference could be observed between binding at 04°C and at 37°C. This phenomenon was explained in terms of a rapid non-specific adsorption of transferrin at W"C, which takes place especially at higher transferrin concentrations, and a specific binding of transferrin at 37°C observed presumably at low concentrations. 3. The maximum number of specific transferrin-binding sites was found to be approximately 190000 sites per rat reticulocyte and 330000 sites per nucleated rat bone-marrow cell. The latter number corresponds to 500 000700 OOO sites per nucleated erythroid cell. 4. It was concluded that maturation of the erythroid cell is accompanied with a progressive loss of transferrin binding sites on the cell membrane. 5. When bone-marrow cells obtained after incubation with doubly-labelled transferrin were lysed with distilled water or with the detergent Nonidet P-40, differences in the

Key words: bone-marrow cells, iron uptake, reticulocytes, transferrin.

Introduction The iron required for haemoglobin synthesis by immature erythrocytes is provided by the plasma, where it is bound to transferrin. Each molecule of transferrin is capable of binding a maximum of two iron atoms. The mechanism by which iron is taken up by immature erythroid cells and transported to the haemsynthesizingmitochondria is poorly understood (seereviews by Bezkorovainy & Zschocke, 1974; Zschocke & Bezkorovainy, 1974). The initial step of this process was found to be the association of iron-transferrin with the cell membrane (Jandl, Inman, Simmons & Allen, 1959; Jandl & Katz, 1963; Baker & Morgan, 1969a, b, 1971). It has been postulated by Jandl and coworkers that immature erythroid cells bear specific binding sites for transferrin (Jandl et al., 1959; Jandl & Katz, 1963). Once bound to these receptor sites, transferrin releases its iron, which is subsequently transported to the mitochondria via intracellular intermediates. In support of this hypothesis are the findings that erythroid

Correspondence: Dr N. J. Verhoef, Department of Chemical Pathology, Faculty of Medicine, Erasmus University Rotterdam, Rotterdam, The Netherlands.

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N. J , Verhoef and P. J. Noordeloos

cell lysates contain iron-containing compounds other than transferrin (Zail, Charlton, Torrance & Bothwell, 1964;Primosigh & Thomas, 1968; Borovi, Poiika & Neuwirt, 1973;Workman & Bates, 1974) and that only part of the 59Fe recovered in the stroma fraction of cell lysates, after incubation of cells with doubly-labelled transferrin, appeared to be bound to transferrin (Garrett, Garrett & Archdeacon, 1973;Speyer & Fielding, 1974; Fielding & Speyer, 1974). Most of these studies were performed with reticulocytes. Our aim was to investigate the mechanism of iron uptake by developing erythroid cells more immature than reticulocytes as the rate of haemoglobin synthesis reaches maximum values while the immature cells are still nucleated (Lajtha & Suit, 1955; Najean, Donio & Dresch, 1969; Turpin, 1970). The interaction between transferrin and rat bone-marrow cells has been shown to be species-specific (Verhoef, Kremers & Leijnse, 1973a). Bovine transferrin, for example, appeared to be a very poor transmitter of iron to rat bone-marrow cells, which could be ascribed to its lower affinity for receptor sites on these cells (N. J. Verhoef, H. C. M. Kester & P. J. Noordeloos, unpublished work). This point is of some consequence as foetal bovine serum is added to the bone-marrow cell cultures used for the study of the interaction between transferrin and these cells. A difference in affinity for receptor sites on rat bone-marrow cells was also found in the work referred to between rat apotransferrin and monoferric- or diferrictransferrin. In the present study experiments are described on the binding of transferrin to rat bone-marrow cells and rat reticulocytes to estimate the number of binding sites on the respective cells and the association constants for the interaction between transferrin and these cells. In addition, someexperiments aredescribed in which the fate of transferrin subsequent to its binding to the cell membrane was studied.

Materials and methods Animals and general methods Male Wistar rats, 12-16 weeks of age, were reared on a standard laboratory diet (Hope Farms, Woerden, The Netherlands) containing 132 mg. of iron/kg. Some haematological variables are summarized in Table 1. The

TABLE 1. Some haeniatological and chemical data of rat blood

n = Number of rats.

Value Haemoglobin (mmol of Fe2+/l) Packed cell volume (1/1) Erythrocytes (10’’ x no./l) Leucocytes (lo9 x no./l) Mean corpuscular volume (fl) Mean corpuscular haemoglobin (amol) Mean corpuscular haemoglobin concn. (mmol of Fe2+/1) Serum osmolality (mosmol/kg) Serum iron (pnol/l) Total iron-binding capacity (wnol/l) Iron saturation (%)

n

Mean+s~

66 164 1 I2 107 83

9.7+ 1.3 0.46+ 0.03 6.6+ 1.2 9.9& 2.9 67+ 12

43

1310k 199

60 49 38

21.3k2.8 300+ 8 32.5+ 6.4

32 32

84+ 16 40+ 10

haemoglobin concentration was determined according to Van Kampen & Zijlstra (1961)and the packed cell volume according to Dacie & Lewis (1970) with the use of micro-capillary tubes. The serum iron concentration and totaliron-binding capacity were estimated by the use of sulphonated bathophenanthroline according to modified methods of Trinder (1956) and Ramsay (1957) respectively. Serum osmolality was determined with an Advanced Osmometer model 31 LAS (Newton Highland, Mass., U.S.A.) and cells were counted with an electronic cell counter (MCC-1002B ,Toa Electric, Kobe, Japan). Radioactivity was measured in a scintillation spectrometer (Packard model 5220. Packard Instruments, Downers Grove, Ill., U.S.A.). Minimal essential medium with Hanks’ salts and 2-(N-2-hydroxyethylpiperazinN’-y1)ethanesulphonicacid (Hepes buffer), phosphate-buffered sodium chloride solution according to Dulbecco & Vogt (1954), Hanks’ balanced salt solution, penicillin and streptomycin and foetal bovine and calf sera were purchased from Flow Laboratories, Irvine, Scotland. Garamycin (gentamycin sulphate) was obtained from the Schering Corp., Bloomfield, N.Y., U.S.A. Transferrin Preparation. Rat transferrin was prepared in this laboratory by Mr W. L. van Noort, as described by Verhoef & Van Eijk (1975).

Tramferrin and erythroid cells Radioisotope-labelled transferrin. "Fe was obtained as sterile ferric citrate, of specific radioactivity 5-20 mCi/mg of Fe, and lZ5Ias sodium iodide, carrier-free and free from reducing agents, from The Radiochemical Centre, Amersham, Bucks., U.K. Iron-free transferrin, prepared as described previously (Verhoef et al., 1973a), was iodinated according to a procedure based on that of Hunter & Greenwood (1962) as follows: 12'1 was diluted with lZ7I(as NaI) to give after iodination approximately 1 molecule of iodine/ molecule of transferrin. The diluted I2'I solution was added to transferrin, solubilized in a phosphate buffer (100 mmol/l, pH 7.5). The transferrin concentration in the reaction mixture was approximately 20 mg/ml. To start the reaction, 100 pg of chloramine-T (BDH Chemicals, Poole, Dorset, U.K.) in 10 p1 of phosphate buffer was added to 1 ml of the reaction mixture. After 15 min at room temperature the iodine still present was reduced by addition of 100 pg of sodium metabisulphite (Merck, Darmstadt, Germany) in 10 pl of phosphate buffer. The reaction mixture was subsequently applied to an ion-exchange column (8 cmxO.5 cm) of Amberlite IRA-400 (Cl -) (BDH Chemicals) to remove unbound iodine. Transferrin was eluted with phosphate buffer (100 mmol/l, pH 7.5). Fractions (10 drops) were collected and their radioactivities estimated. The radioactive peak fractions were pooled and 1251-labelled transferrin was dialysed against several changes of distilled water and thereafter freeze-dried. Iodination efficiency was 50-70%. To prepare doubly-labelled transferrin, l 2'Ilabelled transferrin was solubilized in phosphatebuffered NaCl solution, the solution was filtered through a Gelman filter (0.22 pm) and [sqFe]ferriccitrate was added. The mixture was incubated at 37°C for 1 h. Transferrin concentrations were measured by the absorbence at 280 nm with E i 5 = 11.3 (Verhoef & Van Eijk, 1975). Iron saturation is given in the legends to Figures. Cell suspensions

Reticulocytosis was produced in rats by removal of 5 ml of blood each day on 5 days, with a 3 day interval between the thud and fourth specimens. The packed cell volume of the reticulocyte-rich blood used in the various

89

experiments varied from 15 to 22% and the reticulocyte count from 14 to 70%. The blood was collected into heparinized tubes, and the cells were washed with cold phosphate-buffered NaCl solution. Rat bone-marrow cells were isolated as described previously (Verhoef et al., 1973a). Differential nucleated cell counts of the cell suspensions were carried out on dried cell smears stained with May-Griinwald-Giemsa stains. Two thousand cells were examined. The mean percentages for the immature erythroid cells were: proerythroblasts 0.6%, erythroblasts 31.8% and normoblasts 9.4%. The ratio of myeloid cells to nucleated erythroid cells wasapproximately1.14:l.Thesefiguresagreewell with those obtained by others for rat bonemarrow-cell suspensions (Harris & Burke, 1957; Hulse, 1964). Zncubation experiments

Incubation was carried out in 35 mm x 10 mm plastic tissue culture dishes (Falcon Plastics, Los Angela, U.S.A.) at 37°C in an atmosphere of air/COz (95: 5). A typical incubation mixture (1.5 ml) contained 1-15 x lo7 reticulocytes or 1.5 x lo7 nucleated bone-marrow cellslml in a medium consisting of minimum essential medium with Hanks' salts and Hepes buffer (20 mmol/l), supplemented with foetal bovine serum (lo%, v/v), sodium bicarbonate (final concentration 9 mmol/l) and penicillin, streptomycin and garamycin to final concentrations of 100 i.u., 100 i.u. and 0.16 mglml respectively. The amount of doubly-labelled transferrin to the cells is given in the legends to Figures. After incubation at 37°C for 3 h (unless otherwise indicated) the cells were harvested and washed at 0 4 ° C with cold Hanks' balanced salt solution containing Hepes buffer (10 mmol/l) and 100 i.u. each of penicillin and streptomycin/ ml. Subsequentlythe radioactivity of the washed cells was estimated. Where indicated cells were lysed by the addition of 1.0 ml of distilled water at M " C , after thoroughly mixing followed by 0.1 ml of 10% NaCl or by the addition of 1.0 ml of 1 % (v/v) Nonidet P-40 (Shell Chemie, Rotterdam, The Netherlands) in phosphate-buffered NaCl solution. Membranes were removed by centrifugation at 15 OOO g in a Sorvall RC2-B centrifuge at 4°C for 15 min and washed with NaCl solution. From the combined super-

N. J. Verhoef and P. J. Noordeloos

90

natants haem iron was extracted according to a slightly modified procedure of Thunell (1965). Finally the radioactivity of the membrane, haem iron and non-haem iron fractions was determined.

25 -

a

Results

20-

g

0

Binding of transferrin and uptake of iron

In preliminary experiments, the binding of * 3sI-labelled transferrin to rat bone-marrow cells was studied in mixtures containing 2 nmol of transferrin and 15 x lo6 nucleated cells/ ml. Under these conditions transferrin appeared to be the limiting factor in the iron uptake (Fig.

9

5

0

15-

r 0) c

5

10-

1). In using a similar ratio between doubly-

labelled transferrin and nucleated cells, no timedependence of * sI-labelled transferrin binding to cells could be detected, although the iron uptake increases linearly with time (Fig. 2a). Comparable results were recorded with rat reticulocytes instead of rat bone-marrow cells

0

I

2

Concn. of transfwrin (nmol/ml)

FIG. 1. Effect of the transferrin concentration on the uptake of s9Fe. Transferrin saturation was 20%. Results are expressed as pmol of "Fe taken up by bone-marrow cells per incubation mixture.

i

(a)

EC

-- 6 c 0

-aE 9

Q

: 4c

.x 0 n

3

2c

0

I

I

10

20

'

30 + -/

Incubation time (mid

3

Incubationtime (mid

FIG.2. Effect of the time of incubation on the binding of '2sI-labelled transferrin and uptake of 59Fe. Doubly-labelled transferrin, saturated to 50% with iron, was added to a final concentration of 2 nmol/ ml (a) or 0 2 5 mmol/ml (b). Zero points of time were obtained by adding ice-cold doubly-labelled transferrin to cell suspensions at 04"C, immediately followed by harvesting and washing the cells by centrifugation at 04°C. Results are expressed as pmol of "Fe taken up and 12sI-labelled transferrin bound by bone-marrow cells per incubation mixture. 0 , Binding of lZsI-labelled transferrin; 0 , uptake of "Fe.

91

Transferrin and erythroid cells

(Verhoef, Kremers & Leijnse, 1973b). Apparently, a rapid non-specific adsorption of transferrin to the cells occurs at W C , which overshadows a possible specific binding at higher temperatures. At much lower concentrations of transferrin, e.g. 0.25 nmol/ml, however, a time-dependence of transferrin binding could be observed (Fig. 2b), indicating that another type of interaction, which is dependent on incubation at higher temperatures, becomes visible. Iron uptake starts from a very low value and is nearly linear during an incubation time of at least 3 h. By contrast, 1z51-labelled transferrin binding reaches maximum values within the first 10 min of incubation.

0

I

I

1

I

l

2

3

4

The pattern of iron uptake was comparable with that presented in Fig. 1. From the Z51-labelled transferrin-binding curve the number of binding sites per cell may be calculated according to the method of Scatchard (Weder, Schildknecht, Lutz & Kesselring, 1974). Theoretically, the Scatchard plot for a binding reaction involving two sorts of binding sites is not a straight line, which makes it difficult to calculate directly the number of specific binding sites. However, the number of non-specific binding sites seems to be much higher than the number of specific binding sites as the second phase of 251-labelledtransferrin binding (Fig. 3) is linear up to at least 3 nmol/ml. Similar results were found by Baker & Morgan (1969a) for transferrin binding to rabbit reticulocytes. Notwithstanding the high number of nonspecific, binding sites, specific binding could be observed at low transferrin concentrations, which indicated that the association constant for the interaction between transferrin and specific binding sites is much higher than for the non-specific interaction. These considerations lead to the conclusion that at low transferrin concentrations the experimental Scatchard plot would approximate a linear curve corresponding to the curve for specific binding (Weder et al., 1974). In Fig. 4 the results of two experiments are presented. The number of specific binding

Cwrnof tmmferrin ~ d / m l )

FIG. 3. Effect of the transferrin concentration on the binding of 1251~labelled transferrin and uptake of 59Fe. Transferrin saturation was 30%. Results are expressed as pmol of 59Fe taken up and 12sI-labelled transferrin bound by bone-marrow cells per incubation mixture. 0 , Binding of 1Z51-labelledtransferrin; 0, uptake of 59Fe.

Fig. 3 presents the effect of varying the transferrin concentration on the '251-labelled transferrin binding and uptake of s9Fe. After an initial steep increase in lZ5Ibinding with increasing transferrin concentrations a second more gradual increase occurs. This pattern strongly suggests a non-specific adsorption of IZsIlabelled transferrin to rat bone-marrow cells at higher transferrin concentrations, a phenomenon which was also observed with rabbit reticulocytes (Baker & Morgan, 1969a, 1971).

icsr( m o l e c u l a s / ~ a t e d c d FIG.4. Transferrin binding by bone-marrow cells plotted according to the Scatchard method. Results of two experiments are plotted. r = number of transferrin molecules bound per nucleated cell. Tr = molar concentration of unbound transferrin. Extrapolation to the abscissa gives n, the maximal number of specific binding sites per nucleated cell. The slope of the curve equals the negative value of the association constant K.

N. J. Verhoef and P . J. Noordeloos

92

sites calculated by extrapolating the lines to the abscissa were found to be 280 OOO and 310 OOO per nucleated cell respectively. These figures represent the maximum number of binding sites as even at the low transferrin concentrations applied, small amounts of non-specific binding probably occurs. The intrinsic association constant K equals the negative value of the slope of the curves in Fig. 4. In the experiments presented K values were obtained of 4.8 x lo6 and 2.2 x lo7 1. mol-' respectively. For the same reason as the calculated number of transferrin-binding sites are maximal values, the calculated K values are minimal values. TABLE 2. Transferrin-bindingcharacteristics of rat bonemarrow cells and rat reticulocytes

Mean resultsf so are given for five experiments, with the range in parentheses. n = maximum number of binding sites per nucleated cell o r reticulocyte.

Bone-marrow cells 330 OOO+ 67 OOO (260000-430 OOO) Reticulocytes 189 OOOk 96 OOO (85 000-343 OOO)

2.5k 1.4 (1.1-4.8) 4.1 f 3.5 (09-8.3)

A summary of the results obtained is given in Table 2. The number of specific binding sites per nucleated cell was in the range 260000430000 in five different experiments, and K values varied in these experiments from 1.1 to 4.8 x lo6 1. mol-I. It is unlikely that these

results are influenced by the presence of foetal bovine transferrin in the incubation mixture as bovine transferrin does not inhibit the iron uptake from iron bound to rat transferrin under the present conditions (unpublished work). Iron saturation of * 51-labelled transferrin used in these experiments varied from 20 to 50%. No correlation between iron saturation and the number of transferrin-binding sites or the association constant could be detected. This agrees with the finding that 12sII-labelledtransferrin binding to rat bone-marrow cells varied only slightly with iron saturation (unpublished work). As it is generally accepted that the number of specific binding sites per cell diminishes during maturation (Jandle & Katz, 1963; Kornfeld, 1969) it seemed of interest to compare the transferrin-binding characteristics to cell receptors on bone-marrow cells with those on reticulocytes. For this purpose rat reticulocyte-rich blood was used (see the Materials and methods section). In the same way as described above for bone-marrow cells, transferrin binding to reticulocytes was studied. The doubly-labelled transferrin was saturated with 59Feto 40%. The number of binding sites and the association constant calculated from these experiments are also given in Table 2. Subcellular distribution of 251-labelledtransferrin and Fe

In previous experiments (Verhoef et ol., 1973a) bone-marrow-cell lysates were prepared after lysis with distilled water followed by washing the membranes with distilled water.

TABLE 3. Recovery of 59Fe in different subcellular fractions after haemolysis of bone-marrow cells by various methods Results are expressed as percentages of the radioactivity recovered in cell lysates. The absorbence at 380 nm of the haemin fraction is a quantitative measure for the total (labelled and non-labelled) haemin concentration (Thunell, 1965).

Procedure Distilled water Distilled water+ NaCI* Tris buffer, pH 8.15 Nonidet P-40 (I%)?

Membrane fraction

Haemin fraction

Non-haemin fraction

(%)

(%)

(%)

16.6 12.7 9.6 5.2

59.4 67.0 63.3 74.4

20.0 21.4 23.8 19.4

Recovery

(%)

E380 of haemin fraction

96.0 101.1 96.7 99.0

0.560 0.769 0.805 0.825

* After lysis with distilled water 0 1 vol of 10% (w/v) NaCl was added. t In phosphate-buffered NaCl solution.

Transferrin and erythroid cells

This procedure results in a more or less redcoloured membrane fraction, indicating that some haemoglobin precipitates under these conditions (cf. Hrinda & Goldwasser, 1969). To investigatewhether other methods of haemolysis prevent the precipitation of haemoglobin a number of methods were applied to bonemarrow cells incubated in the presence of 59Fe bound to rat transferrin. After removal of the membranes haemin was extracted from the supernatant as described in the Materials and methods section. In all procedures applied the majority of the radioactivity could be recovered in the haemin fraction, indicating that the larger part of the iron taken up by the cells was incorporated into haem (Table 3). The highest amount of haemin could be extracted from the supernatant after lysis with 1 % Nonidet P-40 in phosphate-buffered NaCl solution. Under these conditions the membranes were virtually colourless, indicating that no haemoglobin precipitated.

TABLE 4. Fractionation of the 59Fe and 1251-labelled transferrin-containing membrane fraction The washed membrane fraction, obtained from cells incubated with doubly-labelled transferrin and lysed with distilled water, was subjected to treatment with Nonidet P-40 (1% in phosphate-bufferedNaCl solution). The mixture was again fractionated into three fractions and the distribution of 59Feand 1251-labelledtransferrin about these fractions was determined. Results are presented as pmol of 59Fe and 1251-labelledtransferrin recovered into each fraction. Membrane Haernin Non-haemin fraction fraction fraction (Nonidet) 59Fe (pmol) 1251-labelledtransferrin (pmol)

47 1 .o

22 0.1

14

4.1

In the following experiment rat bone-marrow cells were incubated in the presence of doublylabelled transferrin and thereafter Iysed with distilled water followed by the addition of 0.1 vol. of 10% NaCl. The washed membrane fraction was isolated and subjected to treatment with 1% Nonidet P-40 in phosphate-buffered NaCl solution, whereafter the mixture was again fractionated into a membrane, a haemin

93

and a non-haem fraction. As may be seen from Table 4, the membrane fraction obtained after lysis with distilled water contains appreciable amounts of [’ 9Fe]haemin. Furthermore, even the membrane fraction obtained after treatment with Nonidet P-40 contains much more 59Fe than 251-labelledtransferrin, indicating that the majority of the 59Fein this fraction is not bound to * 251-labelledtransferrin. The same conclusion could be drawn for the non-haem fraction. Essentially the same results were obtained by incubating rat bone-marrow cells with doubly-labelled human or rabbit transferrin. Both species of transferrin are capable of transferring iron to rat bone-marrow cells, although to a lesser extent than rat transferrin (Verhoef et al., 1973a). Discussion Binding of transferrin and uptake of iron

The kinetics of binding of * 251-labelled transferrin and uptake of 59Fe by rat bonemarrow cells at relatively low concentrations of transferrin are comparable with those by human reticulocytes (Jandl & Katz, 1963; Edwards & Fielding, 1971), rabbit reticulocytes (Morgan & Laurell, 1963; Baker & Morgan, 1969a, 1971) and rabbit bonemarrow cells (Kailis & Morgan, 1974). At higher transferrin concentrations binding of transferrin already reaches maximal values at 0°C. Probably this represents a non-specificadsorption to bone-marrow cells, as also occurs to rabbit reticulocytes (Morgan & Laurell, 1963). The transferrin concentration was found to be the limiting factor for iron uptake even at concentrations of 2 nmol of transferrinlml. This suggests that under these conditions non-specific adsorption occurs, although not all specific binding sites on the cell membrane are occupied by transferrin. Only at concentrations lower than approximately 1 nmol of transferrin/mlwere no indications for a non-specific binding after incubation at 37°C obtained under the conditions used. Workman, Graham & Bates (1975) have described experiments from which it was concluded that the chloramine-.r method for iodinating transferrin leads to non-specific binding of transferrin to reticulocytes. However, the experiments were carried out at high transferrin concentrations, namely 5 x mol/l, which is about 50 times

94

N . J. Verhoef and P. J. Noordeloos

the concentration critical for non-specific binding as found in the present study (Fig. 3). Differences in the kinetics of transferrin binding at different transferrin concentrations were also observed by Lane (1972, 1973), who studied the binding of human transferrin to rabbit reticulocytes. The maximum number of specific transferrinbinding sites on rat reticulocytes appeared to be in the range 85000-343000, mean value 189 OOO (Table 2). These figures are somewhat lower than those reported by Baker & Morgan (1969a) for the number of transferrin-binding sites on rabbit reticulocytes. The latter authors estimated a mean number of 300 OOO sites per reticulocyte with a range of 200 000-560 OOO. A much lower number of sites on rabbit reticulocytes was found by Kornfeld (1969), namely 26 000-45 OOO. Possibly, the last figures are less acceptable since human transferrin rather than rabbit transferrin was used to study the binding sites on rabbit reticulocytes. A number of 50 OOO transferrin-binding sites was estimated for human reticulocytes (Jandl & Katz, 1963). The number of specific receptor sites on rat bonemarrow cells was found to be in the range 260000-430000 per nucleated cell, mean number 330 OOO (Table 2). As nucleated bonemarrow cells include a number of non-erythroid cells which do not assimilate iron (Lajtha & Suit, 1955; Labardini, Papayannopoulou, Cook, Adamson, Woodson, Eschbach, Hillman & Finch, 1973) and presumably do not bind transferrin, the number of binding sites on nucleated erythroid cells should be higher. The bone-marrow-cell suspensions used in the present studies contained about 42% erythroid cells with a myeloid to erythroid cell ratio about 1.14:l. Similar values are reported by others (Harris & Burke, 1957; Hulse, 1964). Taking into account that the ratio of nucleated erythroid cells to reticulocytes in rat bone marrow was found tobe5:l to lO:l,the numberoftransferrin receptor sites on rat nucleated erythroid cells may be estimated to be in the order of 700 OOO, which is more than three times the number on rat reticulocytes. As the mean surface area of nucleated erythroid cells was calculated by Kailis & Morgan (1974) to be approximately twice that of reticulocytes, the number of specific binding sites per unit of surface area on nucleated cells seems to be higher than that on reticulocytes.

Obviously, one consequence of cell maturation is the loss of receptor sites on the cell membrane, as has been suggested by others (Jandl et nl., 1959; Jandl & Karz, 1963). At least partly this loss can be explained by the smaller size and surface area of reticulocytes. The intrinsic association constant for the binding of transferrin to specific receptor sites was found to be approximately the same for rat bone-marrow cells and rat reticulocytes (Table 2). The saturation degree of transferrin samples used for the experiments varied between 20 and 50%. One may argue that the association constant possibly depends upon the saturation degree of transferrin, as some authors reported that monoferric-transferrin is bound more weakly to reticulocytes than diferric-transferrin, although this has been challenged by others (Fletcher, 1969; Kornfeld, 1969; Lane, 1973; Harris & Aisen, 1975). However, transferrin binding to rat bone-marrow cells was found to vary only slightly with the iron saturation; moreover we have found that the binding of monoferric- and diferric-transferrin to rat bone-marrow cells appeared to be very similar. For the transferrin binding to rabbit reticulocytes an average association constant of 200 OOO 1. mol-' was found (Baker & Morgan, 1969a), which is much lower than the values calculated in the present study for rat reticulocytes. Further experiments are required to investigate whether these differences reflect a real species difference between the reticulocytes or whether differences in experimental conditions are reponsible for this discrepancy. It has to be noted that in the present study, as well as in that of others, reticulocytes obtained after stimulated erythropoiesis rather than normal reticulocytes were used. These reticulocytes are of excessive size and should undergo shrinkage by loss of substantial amounts of their plasma-membrane lipids and both cell water and some haemoglobin (Brecher & Stohlman, 1961; Ganzoni, Hillman & Finch, 1969; Shattil & Cooper, 1972; Come, Shohet & Robinson, 1972, 1974). Moreover, these cells have abnormal membrane surface characteristics as compared with normal reticulocytes (Walter, Miller, Krob & Ascher, 1972). It cannot be excluded, therefore, that the results obtained with these macroreticulocytes are not representative for those which would be obtained with normal reticulocytes.

Transferrin and erythroid cells Mechanism of iron uptake From the results presented in the present paper it may be concluded that the membrane fraction obtained after lysis of rat bone-marrow cells incubated with doubly-labelled transferrin, contains appreciable amounts of 59Fe not bound to transferrin. These results support the model originally proposed by Jandl et al. (1959) and Jandl & Katz (1963), that the binding of the iron-transferrin complex to erythroid cell receptors is followed by the dissociation of the complex and the transport of iron to the mitochondria by means of intracellular intermediates. Part of the iron recovered in the membrane fraction is presumably bound to one or more of such intermediates. The presence in membrane fractions of iron bound to components other than transferrin could also be established in membrane fractions of rabbit reticulocytes(Garrett et al., 1973) and of human reticulocytes (Speyer & Fielding, 1974; Fielding & Speyer, 1974). Moreover, anumber of authors did observe a radioactive labelled iron-containing component with a low molecular weight present in the cytosol of reticulocytes and of bone-marrow cells after incubating these cells with radioactive iron bound to transferrin (Zail et al., 1964; Primosign & Thomas, 1968; Borovh et al., 1973; Speyer & Fielding, 1974; Workman & Bates, 1974). All these results are strongly in favour of the model of Jandl and not of the model proposed by Morgan that the transferrin-iron complex enters the cell and is again released by endocytosis and exocytosis respectively. The latter model is based upon the finding that SI-labelled transferrin could be detected within reticulocytes by means of autoradiography (Morgan & Appleton, 1969) and that the cytosol of reticulocytes seemed to contain 2sI-labelledtransferrin after incubation of cells with zsI-labelled transferrin (MartinezMedellin & Schulman, 1972; Sly, Grohlich & Bezkorovainy, 1975). Particularly the latter finding is questionable as it could not be excluded that during the lysis procedure of cells some transferrin bound to the membrane is lost and thus recovered in the cytosol fraction. In the experiments presented in the present paper also some * Wabelled transferrin could be detected in the non-haem fraction (Table 4). However, the finding that the membrane fraction contained appreciable amounts of sgFe not

95

bound to transferrin was thought to be of greater value than the small amounts of radioactive transferrin recovered in the non-haem fraction. More detailed studies are required, however, to analyse the source of 12sI-labelled transferrin found in the latter fraction.

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