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The mobilisation of iron from the cell stroma during erythroid maturation
Vol. 61, No. 1,1974
BIOCHEMICAL
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
THEMOBILISATION OF IRON FROMTHECELL STROMADURINGERYTHROID MATURATION M.J. Denton,* H.T. Delves*
and H.R.V. Arnstein*
*Department of Biochemistry, University of London King's College, Strand, London WC2R2LS, England *Institute of Child Health, 30 Guilford London WClN lEH, England Received
September
Street,
9,1974
SUMMARY: Adult mammalianerythroblssts from an enaemic rabbit were separated -actions of cells at different stages of development using the velocity sedimentation technique. The iron content of the stroma and cytoplasm of the cells in each fraction was determined.byatomic. absorption spectroscopy. A high proportion of the iron in the dividing erythroblasts was found to be associated with the cell stroma. After the final cell division the proportion of stromal iron rapidly declines ana it appears that stromal iron is mobilised at this stage and utilised by the non-dividing erythroblasts for haemoglobin synthesis.
INTRODUCTION Iron incorporation synthesis.
by ery-throid cells is not directly
Erythroblasts
continue to incorporate
of haemis blocked by lead administration erythroblasts
iron even when the synthesis In the immature basophilic
before active haemoglobin synthesis has commencediron incorporation
has been shownby autoradiography
(2) and'by "Fe uptake (3) to be at least as
rapid as in the more mature erythroblasts In previous
(1).
related to haemoglobin
studies
we have demanstrated that the intensive
synthesis occurs only after
the erythroid
an& entered the non-dividing considerable mobilisation ment coincidentally
in which haemoglobin synthesis is active. phase af haemoglobin
cell has undergone the final
compartment (4).
cell division
Here we report evidence that a
of stromal bound iron occurs in the non-dividing
with the intensive
compart-
phase of haemoglobin synthesis.
EXPERIMENTAL Cell Separation New Zealsna &.$te rabbits of neutralised
(2 kg) vrere made anaemic by five
phenylhydrazine HCl (5).
Copyright 0 1974 by Academic Press, Inc. All rights of reproduction in any form reserved.
Two days after
8
daily injections
the cessation of
VoL61,No.
BIOCHEMICAL
1,1974
the animals were killed
injections
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
and an erythroid
bone mruTOW
SUSPelEd.on
me bone marrow suspension was then subjected to fractionation
prepared. the velocity
sedimentation technique essentially
as previously
w%
by
described (6).
A square Perspex sedimentation chamber of cross section 2500 cm2 was used an& 30 fractions
of 250 ml each were collected
The sedimentation velocity the fact that at unit gravity to its size,
sedimentation.
technique separates ery-throid celis by exploiting the sedimentation velocity
and during ery-throid
in size from the
15
reticulocytes.
Fractions 1 to
fractions
after
differentiation
p diameter basophilic 10
of a cell is proportional
the cells
erythroblasts
cells in fractions
consisted mainly of basophilic ery-throblasts;
fractions
dividing
ery-throblasts,
compartment.
20 to 25 of The
because at the final
to and immediately following
cell division
and during subsequent maturation to the reticulocyte
those in fractions
Using this technique complete
separation was achieved between cells immediately prior cell division
fractions
25 to 30 mainly of reticulocytes.
1 to 20 were actively
21 to 30 belonged to the non-dividing
the final
decrease
to the 8 pm diameter
10 to 20 mainly of polychromatio erythroblasts;
orthochromatic erythroblasts;
continually
the cell size is halved
stage the cell size continues
to decline graaually. Iron Determinations The cells in each fraction centrifuged containing 0.1
from the sedimentation chamber were
and washed three times in phosphate-buffered saline (P.B.S.) 8 g. N&l,
0.2 g. KCl, I.15 g. NapOh,
g. M&12EB20 per like
fraction
collected
of solution.
0.2 g. KH2P04, 0.1 Q. caC12,
A small aliquot
of cells from each
was removed, and the number of cells in each fraction
a hsematooytometer. parts auuble distilled
determined using
The Cells were then lysed by hype-osmotic shock using 5 water to 1 part of cells.
The lysates were centrifuged
at 20000 g* for 10 min. to separate the stroma from the c.@oplasm. pellets
were washed twice in distilled
ti@pt pellet.
(pH 7.4)
The supernatant fluid
water sn& finally
!l!he stromal
centrifu@;ed into a
remaining from each wa,shw&8 added to the
corresponding SUpernatant cytoplasmic fraction.
9
Allremaining
fhia~s
carefully
Vol. 61, No. 1,1974
removed from the pellet of cone. nitric After
BIOCHEMICAL
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
using a Pasteur pipette.
acid (Aristar
grade, British
To each pellet
Drug Houses Ltd.,
was added 150 ~1
Poole, Dorset).
about 6 hr. at 2O'C the pellets had been completely digested. !Chenitric
acid solutions were dilutea
to a volume of 1.0 ml with distilled
content of each of the diluted
The iron
digests was determined by atomic absorption using
a Perkin Elmer atomic absorption spectrometer Moael 303. in the distilled
water.
The concentration
of iron
water and in the P.B.S. was also determined.
The cytoplasmic lysates were concentrated to a volume of 0.75 ml by vacuum desiccation.
The iron content of each lysate was then determined by atomic
absorption spectroscopy.
The absorbance at 415 nm of each lysate was also
m-ma. RESULTS The iron content of the distilled The number of cells in each fraction
water and of the P.B.S. was negligible. is shown in Fig. 1.
'phe cell types sedi-
menting in the major regions of the sedimentation gradient are indicated. total
iron content of the aeveloping erythroblast
continuously throughout erythropoiesis the basophilic erythroblasts
The
would appear to decrease
from a level of approx. 5 pg. per cell in
to approx. 0.7 pg. in the maxrow reticulocytes
The iron content of the polychromatic erythroblasts
FRACTION
in fractions
15-18
is about
NUMBER
1. Cell sedimentation profile and total fractionated by velocity sedimentation. l cell number in each fraction A iron content of each fraction A, basophilic cells; B, polychromatic cells; D, reticulocytes. Fig.
10
iron content of cells
C, orthochromatic
(Fig. 1).
cells;
Vol. 61, No. 1,1974
BIOCHEMICAL
1.3 pg. per cell.
maturation, fractions
AND BIOPHYSICAL
These cells unaergo the final
giving rise to the non-dividing
half that of their
polychromatic
cell division
of erythroid
orthochromatic ergrthroblasts in
with an iron content of approx.
21-25
RESEARCH COMMUNICATIONS
precursors.
0.7
pg. per cell,
Subsequently, their
which is about iron content remains
almost constant during loss of the nucleus ana maturation into reticulocyk.es. total
iron content of the dividing
division
ia sufficient
the reticulocyte
ery-throblasts
!Phe
immediately before the final
to accuunt for the iron content of their
cell
daughter cells at
stage without any f'urther uptake of iron.
!Cheiron content of the stroma and cytoplasmic lysate and the absorbance of the cytoplaamio lysate at oy-toplasmic fraction of erythropoiesis cells
415
nm are shown in Fig. 2.
is closely related
The iron content of the
to the absorbance at
stages
A steep increase in the proportion
of cy-toplasmic iron in haemoglobin occurs in the non-dividing compartment about 8% of the total
associated with stromal material. division
nm at all
which suggests that most of the iron in the cytoplasm of ery-throid
is in the form of hsemoglobin.
the dividing
415
After
and passed into the non-dividing
the stromal fraction
compartment.
cell iron is non-haemoglobin
the cells have undergone the final compartment the proportion
declines quite sudaenly to less than 2% of total
FRACTION
In
NUMBER
cell
of iron in cell iron.
Fig. 2. Iron content of cell etroma and cytoplasm. l ,iron content of cell stroma in each fraction; Ir. ,iron content of cell cytoplasm in each fraction; 0 ,absorbsnce at 415 nm of the cytoplasmic lysate from each fraction.
11
Vol. 61, No. 1,1974
The proportion
BIOCHEMICAL
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
of stromal iron continues to decline as the orthochromatic cell
matures into the reticulocy-te. DISCUSSION The fact that the iron content of the maturing erythroblaat
is almost constan+
at a time when haemoglobin synthesis is intense during the period when the cell manufactures most of its
final
haemoglobin complement (4) sug@sts that much of the
iron required for haemoglobin synthesis could be provided for by existing cellular
iron without the necessity for further
iron in the plasma transferrin.
The additional
iron incorporation
intra-
from the bound
fact that a massive shift
in cell
iron from the stroma to cytoplasmic haemoglobin occurs at this time is very strong circumstantial non-dividing
evidence that a major source of iron for haemoglobin synthesis in erythroblasts
is from a pre-existing
iron store in the cell stroma.
The membraneof the red cell has been considered to funotion as an intermediate between the stage of plasma transport iron in ery-throblasts of transferrin-bound
is not clear (7).
A certain proportion
iron on surface receptor sites.
such sites on the reticulooytes although their
and haem synthesis, but the nature of stroma
However there are only 50000
occupying some2% of the cell surface (1, 7) and
numbers are probably waning at this stage it
more than a small percentage of stromal ir& Other membraneiron-binding
could be in the form
is doubtfUl if
anything
could be accounted for in this way.
macromolecular species have been found in retioulocyte
stroma (8) end in addition the stroma of immature ma cells is known to contain iron-rich electron microscopy of ferritin
including reticulocytes
@xnules which have the appearance on light (1, 9).
It is unlikely
and
that muohnon-haemoglobin
iron is haem as the synthesis of haemis closely associated with globin throughout erythropoiesis
(10).
The proportion
of stromal iron in dividing
large and it is tempting to speculate that its supply of iron from becoming rate limiting
erythroblasts fknction
may be to prevent the
during the most active phase of
haemoglobin synthesis in the orthochromatic cells.
12
is surprisingly
Vol.61,No.
1,1974
BIOCHEMICAL
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
REmRENcEs
J.H. (1970) in Regulation of Haematopoiesis, vol. 1, ed. by A.S. Gordon, pp. 539-577, Appleton Century Crofts, New York. Myhre, E. (1964) Scan& J. Clin. Lab. Invest. l6, 201-211. Lajtha, L.G. and Suit, H.D. (1955) Br. J. Haemat. 1, 55-61. Denton, M.J., Spencer, N. and Arnatein, H.R.V. (1974) submitted to Biochem.J. Amstein, H.R.V., Cox, R.A. and Hunt, J.A. (1964) Biochem. J. 22, 648-661. Denton, M.J. and Arnetein, H.R.V. (1973) Br. J. Haemat. 2 , 7-17. Jandl, J.H. and Katz, J.H. (1963) J. Clin. Invest. &, 31 3 -326. Garrett, N.E., Burriss Garrett, R.J. and Archdeacon, J.W. (1973) Biophys. Biochem. Res. Co-. 2, 466-474. Sondhaus, C.A. and Thorell, B. (1960) Blood l.6, 1285-1297. Hodgkin, G. (1970) in Regulation of Haematopoiesis, ~01.1, ed. by A.S. Gordon, pp. 459-469, Appleton Century Crofts, New York.
1. Katz, 2.
3. 4. 5. 6. 7. 8. 9. 10.
13
BIOCHEMICAL
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
THEMOBILISATION OF IRON FROMTHECELL STROMADURINGERYTHROID MATURATION M.J. Denton,* H.T. Delves*
and H.R.V. Arnstein*
*Department of Biochemistry, University of London King's College, Strand, London WC2R2LS, England *Institute of Child Health, 30 Guilford London WClN lEH, England Received
September
Street,
9,1974
SUMMARY: Adult mammalianerythroblssts from an enaemic rabbit were separated -actions of cells at different stages of development using the velocity sedimentation technique. The iron content of the stroma and cytoplasm of the cells in each fraction was determined.byatomic. absorption spectroscopy. A high proportion of the iron in the dividing erythroblasts was found to be associated with the cell stroma. After the final cell division the proportion of stromal iron rapidly declines ana it appears that stromal iron is mobilised at this stage and utilised by the non-dividing erythroblasts for haemoglobin synthesis.
INTRODUCTION Iron incorporation synthesis.
by ery-throid cells is not directly
Erythroblasts
continue to incorporate
of haemis blocked by lead administration erythroblasts
iron even when the synthesis In the immature basophilic
before active haemoglobin synthesis has commencediron incorporation
has been shownby autoradiography
(2) and'by "Fe uptake (3) to be at least as
rapid as in the more mature erythroblasts In previous
(1).
related to haemoglobin
studies
we have demanstrated that the intensive
synthesis occurs only after
the erythroid
an& entered the non-dividing considerable mobilisation ment coincidentally
in which haemoglobin synthesis is active. phase af haemoglobin
cell has undergone the final
compartment (4).
cell division
Here we report evidence that a
of stromal bound iron occurs in the non-dividing
with the intensive
compart-
phase of haemoglobin synthesis.
EXPERIMENTAL Cell Separation New Zealsna &.$te rabbits of neutralised
(2 kg) vrere made anaemic by five
phenylhydrazine HCl (5).
Copyright 0 1974 by Academic Press, Inc. All rights of reproduction in any form reserved.
Two days after
8
daily injections
the cessation of
VoL61,No.
BIOCHEMICAL
1,1974
the animals were killed
injections
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
and an erythroid
bone mruTOW
SUSPelEd.on
me bone marrow suspension was then subjected to fractionation
prepared. the velocity
sedimentation technique essentially
as previously
w%
by
described (6).
A square Perspex sedimentation chamber of cross section 2500 cm2 was used an& 30 fractions
of 250 ml each were collected
The sedimentation velocity the fact that at unit gravity to its size,
sedimentation.
technique separates ery-throid celis by exploiting the sedimentation velocity
and during ery-throid
in size from the
15
reticulocytes.
Fractions 1 to
fractions
after
differentiation
p diameter basophilic 10
of a cell is proportional
the cells
erythroblasts
cells in fractions
consisted mainly of basophilic ery-throblasts;
fractions
dividing
ery-throblasts,
compartment.
20 to 25 of The
because at the final
to and immediately following
cell division
and during subsequent maturation to the reticulocyte
those in fractions
Using this technique complete
separation was achieved between cells immediately prior cell division
fractions
25 to 30 mainly of reticulocytes.
1 to 20 were actively
21 to 30 belonged to the non-dividing
the final
decrease
to the 8 pm diameter
10 to 20 mainly of polychromatio erythroblasts;
orthochromatic erythroblasts;
continually
the cell size is halved
stage the cell size continues
to decline graaually. Iron Determinations The cells in each fraction centrifuged containing 0.1
from the sedimentation chamber were
and washed three times in phosphate-buffered saline (P.B.S.) 8 g. N&l,
0.2 g. KCl, I.15 g. NapOh,
g. M&12EB20 per like
fraction
collected
of solution.
0.2 g. KH2P04, 0.1 Q. caC12,
A small aliquot
of cells from each
was removed, and the number of cells in each fraction
a hsematooytometer. parts auuble distilled
determined using
The Cells were then lysed by hype-osmotic shock using 5 water to 1 part of cells.
The lysates were centrifuged
at 20000 g* for 10 min. to separate the stroma from the c.@oplasm. pellets
were washed twice in distilled
ti@pt pellet.
(pH 7.4)
The supernatant fluid
water sn& finally
!l!he stromal
centrifu@;ed into a
remaining from each wa,shw&8 added to the
corresponding SUpernatant cytoplasmic fraction.
9
Allremaining
fhia~s
carefully
Vol. 61, No. 1,1974
removed from the pellet of cone. nitric After
BIOCHEMICAL
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
using a Pasteur pipette.
acid (Aristar
grade, British
To each pellet
Drug Houses Ltd.,
was added 150 ~1
Poole, Dorset).
about 6 hr. at 2O'C the pellets had been completely digested. !Chenitric
acid solutions were dilutea
to a volume of 1.0 ml with distilled
content of each of the diluted
The iron
digests was determined by atomic absorption using
a Perkin Elmer atomic absorption spectrometer Moael 303. in the distilled
water.
The concentration
of iron
water and in the P.B.S. was also determined.
The cytoplasmic lysates were concentrated to a volume of 0.75 ml by vacuum desiccation.
The iron content of each lysate was then determined by atomic
absorption spectroscopy.
The absorbance at 415 nm of each lysate was also
m-ma. RESULTS The iron content of the distilled The number of cells in each fraction
water and of the P.B.S. was negligible. is shown in Fig. 1.
'phe cell types sedi-
menting in the major regions of the sedimentation gradient are indicated. total
iron content of the aeveloping erythroblast
continuously throughout erythropoiesis the basophilic erythroblasts
The
would appear to decrease
from a level of approx. 5 pg. per cell in
to approx. 0.7 pg. in the maxrow reticulocytes
The iron content of the polychromatic erythroblasts
FRACTION
in fractions
15-18
is about
NUMBER
1. Cell sedimentation profile and total fractionated by velocity sedimentation. l cell number in each fraction A iron content of each fraction A, basophilic cells; B, polychromatic cells; D, reticulocytes. Fig.
10
iron content of cells
C, orthochromatic
(Fig. 1).
cells;
Vol. 61, No. 1,1974
BIOCHEMICAL
1.3 pg. per cell.
maturation, fractions
AND BIOPHYSICAL
These cells unaergo the final
giving rise to the non-dividing
half that of their
polychromatic
cell division
of erythroid
orthochromatic ergrthroblasts in
with an iron content of approx.
21-25
RESEARCH COMMUNICATIONS
precursors.
0.7
pg. per cell,
Subsequently, their
which is about iron content remains
almost constant during loss of the nucleus ana maturation into reticulocyk.es. total
iron content of the dividing
division
ia sufficient
the reticulocyte
ery-throblasts
!Phe
immediately before the final
to accuunt for the iron content of their
cell
daughter cells at
stage without any f'urther uptake of iron.
!Cheiron content of the stroma and cytoplasmic lysate and the absorbance of the cytoplaamio lysate at oy-toplasmic fraction of erythropoiesis cells
415
nm are shown in Fig. 2.
is closely related
The iron content of the
to the absorbance at
stages
A steep increase in the proportion
of cy-toplasmic iron in haemoglobin occurs in the non-dividing compartment about 8% of the total
associated with stromal material. division
nm at all
which suggests that most of the iron in the cytoplasm of ery-throid
is in the form of hsemoglobin.
the dividing
415
After
and passed into the non-dividing
the stromal fraction
compartment.
cell iron is non-haemoglobin
the cells have undergone the final compartment the proportion
declines quite sudaenly to less than 2% of total
FRACTION
In
NUMBER
cell
of iron in cell iron.
Fig. 2. Iron content of cell etroma and cytoplasm. l ,iron content of cell stroma in each fraction; Ir. ,iron content of cell cytoplasm in each fraction; 0 ,absorbsnce at 415 nm of the cytoplasmic lysate from each fraction.
11
Vol. 61, No. 1,1974
The proportion
BIOCHEMICAL
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
of stromal iron continues to decline as the orthochromatic cell
matures into the reticulocy-te. DISCUSSION The fact that the iron content of the maturing erythroblaat
is almost constan+
at a time when haemoglobin synthesis is intense during the period when the cell manufactures most of its
final
haemoglobin complement (4) sug@sts that much of the
iron required for haemoglobin synthesis could be provided for by existing cellular
iron without the necessity for further
iron in the plasma transferrin.
The additional
iron incorporation
intra-
from the bound
fact that a massive shift
in cell
iron from the stroma to cytoplasmic haemoglobin occurs at this time is very strong circumstantial non-dividing
evidence that a major source of iron for haemoglobin synthesis in erythroblasts
is from a pre-existing
iron store in the cell stroma.
The membraneof the red cell has been considered to funotion as an intermediate between the stage of plasma transport iron in ery-throblasts of transferrin-bound
is not clear (7).
A certain proportion
iron on surface receptor sites.
such sites on the reticulooytes although their
and haem synthesis, but the nature of stroma
However there are only 50000
occupying some2% of the cell surface (1, 7) and
numbers are probably waning at this stage it
more than a small percentage of stromal ir& Other membraneiron-binding
could be in the form
is doubtfUl if
anything
could be accounted for in this way.
macromolecular species have been found in retioulocyte
stroma (8) end in addition the stroma of immature ma cells is known to contain iron-rich electron microscopy of ferritin
including reticulocytes
@xnules which have the appearance on light (1, 9).
It is unlikely
and
that muohnon-haemoglobin
iron is haem as the synthesis of haemis closely associated with globin throughout erythropoiesis
(10).
The proportion
of stromal iron in dividing
large and it is tempting to speculate that its supply of iron from becoming rate limiting
erythroblasts fknction
may be to prevent the
during the most active phase of
haemoglobin synthesis in the orthochromatic cells.
12
is surprisingly
Vol.61,No.
1,1974
BIOCHEMICAL
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
REmRENcEs
J.H. (1970) in Regulation of Haematopoiesis, vol. 1, ed. by A.S. Gordon, pp. 539-577, Appleton Century Crofts, New York. Myhre, E. (1964) Scan& J. Clin. Lab. Invest. l6, 201-211. Lajtha, L.G. and Suit, H.D. (1955) Br. J. Haemat. 1, 55-61. Denton, M.J., Spencer, N. and Arnatein, H.R.V. (1974) submitted to Biochem.J. Amstein, H.R.V., Cox, R.A. and Hunt, J.A. (1964) Biochem. J. 22, 648-661. Denton, M.J. and Arnetein, H.R.V. (1973) Br. J. Haemat. 2 , 7-17. Jandl, J.H. and Katz, J.H. (1963) J. Clin. Invest. &, 31 3 -326. Garrett, N.E., Burriss Garrett, R.J. and Archdeacon, J.W. (1973) Biophys. Biochem. Res. Co-. 2, 466-474. Sondhaus, C.A. and Thorell, B. (1960) Blood l.6, 1285-1297. Hodgkin, G. (1970) in Regulation of Haematopoiesis, ~01.1, ed. by A.S. Gordon, pp. 459-469, Appleton Century Crofts, New York.
1. Katz, 2.
3. 4. 5. 6. 7. 8. 9. 10.
13