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.
87
88
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.
Acknowledgments
The authors are indebted to Professor Dr B.
Leijnse for encouragement during the investigations and critical reading of the manuscript.
Miss Evelien van’t Hull (Department of
Pathological Anatomy) is gratefully acknowledged for performing the differential counts on
bone-marrow-cell smears.
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