Báo cáo khoa học: Identification of a 250 kDa putative microtubuleassociated protein as bovine ferritin Evidence for a ferritin–microtubule interaction pdf

Identification of a 250 kDa putative microtubule-
associated protein as bovine ferritin
Evidence for a ferritin–microtubule interaction
Mohammad R. Hasan
1
, Daisuke Morishima
1
, Kyoko Tomita
1
, Miho Katsuki
1
and Susumu Kotani
1,2
1 Department of Bioscience and Bioinformatics, Kyushu Institute of Technology, Iizuka, Fukuoka, Japan
2 Department of Biological Sciences, Faculty of Science, Kanagawa University, Hiratsuka, Kanagawa, Japan
Although microtubules are heteropolymers of a- and
b-tubulin, the diverse roles they play in different cellular
processes, such as cell division, intracellular transport,
cell motility and cytoplasmic morphogenesis, are largely
dependent on various specific binding proteins [1]. These
nontubulin components include the well-known micro-
tubule-associated proteins (MAPs) that coassemble with
tubulin, and are believed to regulate the microtubular
properties in vivo [2]. To date, a considerably large
number of MAPs have been reported, among which, the
brain MAPs, such as MAP1, MAP2 and Tau, were
shown to be responsible for neurite outgrowth in neuron
cells [3,4]. On the other hand, the MAP4 proteins have a
ubiquitous cellular distribution, and have been implica-
ted in the regulation of both cytoplasmic and spindle
microtubules in non-neuronal cells [5,6].

Previously, we reported the presence of MAP4 in
bovine adrenal gland as the major MAP species.
Moreover, an analysis of the adrenal MAPs that coex-
isted with the tubulin after cycles of assembly and dis-
assembly in vitro revealed several minor components in
addition to MAP4 [7]. One of the minor components,
Keywords
ferritin; ferritin–microtubule interaction;
microtubule; microtubule-associated protein
Correspondence
M. R. Hasan, Department of Bioscience and
Bioinformatics, Faculty of Computer Science
and Systems Engineering, Kyushu Institute
of Technology, Iizuka, Fukuoka 820-8502,
Japan
Fax: +81 948 29 7801
Tel: +81 948 29 7840
E-mail:
(Received 22 October 2004, revised 6
December 2004, accepted 8 December
2004)
doi:10.1111/j.1742-4658.2004.04520.x
We reported previously on the purification and partial characterization of
a putative microtubule-associated protein (MAP) from bovine adrenal cor-
tex with an approximate molecular mass of 250 kDa. The protein was
expressed ubiquitously in mammalian tissues, and bound to microtubules
in vitro and in vivo, but failed to promote tubulin polymerization into
microtubules. In the present study, partial amino acid sequencing revealed
that the protein shares an identical primary structure with the widely distri-
buted iron storage protein, ferritin. We also found that the putative MAP

and ferritin are indistinguishable from each other by electrophoretic mobil-
ity, immunological properties and morphological appearance. Moreover,
the putative MAP conserves the iron storage and incorporation properties
of ferritin, confirming that the two are structurally and functionally the
same protein. This fact led us to investigate the interaction of ferritin with
microtubules by direct electron microscopic observations. Ferritin was
bound to microtubules either singly or in the form of large intermolecular
aggregates. We suggest that the formation of intermolecular aggregates
contributes to the intracellular stability of ferritin. The interactions between
ferritin and microtubules observed in this study, in conjunction with the
previous report that the administration of microtubule depolymerizing
drugs increases the serum release of ferritin in rats [Ramm GA, Powell LW
& Halliday JW (1996) J Gastroenterol Hepatol 11, 1072–1078], support the
probable role of microtubules in regulating the intracellular concentration
and release of ferritin under different physiological circumstances.
Abbreviations
MAP, microtubule-associated protein; PVDF, poly(vinylidene difluoride); RB, reassembly buffer; MDBK, Madin–Darby bovine kidney.
822 FEBS Journal 272 (2005) 822–831 ª 2005 FEBS
with an approximate molecular mass of 250 kDa, was
reported as a putative MAP, on the basis of various
properties such as electrophoretic mobility, heat stabil-
ity and immunoreactivity [8]. The protein was found
to be distributed ubiquitously among various bovine
organs. It coassembled with taxol-stabilized micro-
tubules in vitro, and showed some association with the
microtubular network in cultured cells. However,
unlike the other common MAPs (MAP1, MAP2, Tau
and MAP4), the 250 kDa protein lacked the ability to
induce microtubule polymerization from purified tubu-
lin molecules. The molecular shape of the protein, as

determined by electron microscopy, was spherical, in
contrast to the long, rod-like conformations of the
other MAPs [8].
In this study, our attempts to further characterize the
250 kDa protein identified it as the ubiquitous iron stor-
age protein, ferritin, as revealed by comparisons of the
two proteins in terms of their amino acid sequences,
immunoreactivity, molecular mass and shape, and iron
storage ⁄ incorporation properties. As a widely distri-
buted protein among bacteria, plants and animals, fer-
ritin stores iron in the Fe(III) form, preventing the
oxidative damage caused by Fe(II) atoms, and supplies
cells with the necessary iron at an effective concentra-
tion when required. Ferritin has a molecular mass of
450 kDa, and appears as a hollow, roughly spherical
structure with an external diameter of about 12–13 nm.
The inner cavity, which can accommodate up to 4500
Fe(III) atoms, has a diameter of about 8 nm [9–11].
Ferritin is a polymeric protein composed of 24 subunits,
with two subunit types (H and L) in mammals that play
distinct roles in iron homeostasis. The H subunit cata-
lyzes the oxidation of Fe(II), the initial step in the iron
storage process, and the L subunit is known to induce
iron core nucleation [12–14].
Many studies have analyzed ferritin in terms of its
structure, function and regulation, but no report has
established a relationship between ferritin and micro-
tubules. Here, based on microtubule cosedimentation
assays and electron microscopic observations, we
report a novel interaction between ferritin and micro-

tubules in vitro, and hypothesize that microtubules
might be involved in the stability, intracellular pool
and release of ferritin.
Results
Determination of the primary structure
of the 250 kDa protein
Our first attempt to determine the N-terminal amino
acid sequence of the purified 250 kDa protein in its
native form was unsuccessful. Therefore, we thought
that the protein might have chemical modifications at
its N-terminus. Subsequently, we digested the protein
with cyanogen bromide to cleave the protein at methio-
nine residues for internal amino acid sequencing.
Among the digested products, three fragments were
selected and sequenced. The deduced amino acid
sequences were identical to three sequences within the H
subunit of bovine ferritin, as shown by the underlined
sequences (a–c) in Fig. 1, corresponding to residues 39–
53, 72–87, and 102–117, respectively. As expected, all of
the deduced sequences appeared after methionine resi-
dues, suggesting the correctness of the procedures
employed. The results obtained from amino acid
sequencing gave us the first indication that the 250 kDa
protein might actually be ferritin, although none of the
sequences obtained matched the L subunit of ferritin. In
addition, there was a great disparity between the known
molecular mass of ferritin (450 kDa) and the apparent
molecular mass of the 250 kDa protein on SDS⁄ PAGE.
Therefore, further lines of evidence were necessary for a
definitive conclusion.

Comparison of the apparent molecular masses
of ferritin and the 250 kDa protein
To compare the molecular masses of ferritin and the
250 kDa protein, we checked the electrophoretic
mobility of bovine liver ferritin, bovine adrenal cortex
Fig. 1. Amino acid sequence analysis of the 250 kDa protein. A
cyanogen bromide digest of the 250 kDa protein was separated by
SDS ⁄ PAGE, and the fragments were electrophoretically transferred
to a PVDF membrane for internal amino acid sequencing. Three
fragments were selected and sequenced by automated Edman
degradation. The three sequences were identical to three regions
of the ferritin H subunit, as shown by the underlined sequences
(a, b & c).
M. R. Hasan et al. Identification of a putative MAP as ferritin
FEBS Journal 272 (2005) 822–831 ª 2005 FEBS 823
ferritin and the 250 kDa protein by SDS ⁄ PAGE with
or without heat treatment, for the monomeric and
polymeric forms, respectively. As shown in Fig. 2A, all
of the samples showed the same electrophoretic mobil-
ity in either form. This eliminated the confusion about
the molecular mass disparity between ferritin and the
250 kDa protein in the polymeric form. The globular
nature of ferritin in the native form might be a reason
for its faster movement in SDS ⁄ polyacrylamide gels.
With respect to the monomeric forms, it should be
noted here that, unlike other mammalian species where
the molecular masses of the ferritin L and H subunits
are 19 kDa and 21 kDa, respectively, the molecular
mass of the L subunit (20.5 kDa) of bovine ferritin is
larger than that of the H subunit (18.4 kDa), as

observed by SDS ⁄ PAGE. This variation was attributed
to differences in the binding affinity of SDS to the
bovine L chains, rather than any insertions or dele-
tions of amino acids in the bovine ferritin subunits
[15]. We also observed similar properties with the
bovine ferritin subunits in this study. In addition, the
extremely low content of L subunits in adrenal ferritin
and in the 250 kDa protein, as compared to that of
liver ferritin, is also consistent with previous reports
that the H and L subunit contents may differ in mam-
mals, depending on the organs and their iron require-
ments. Again, the abundance of the L subunit in the
liver is important for its iron storage functions [11].
The extremely low abundance of the L subunit in the
250 kDa protein now explains why no sequences
homologous to the L subunit were detected in the
sequencing experiment (Fig. 1).
Immunocrossreactivity of ferritin and the
250 kDa protein
The immunological properties of ferritin and the
250 kDa protein were investigated using an anti-(horse
spleen ferritin) IgG, which recognizes the L subunit of
ferritin, and an anti-(250 kDa protein) Ig. For subunit-
specific detection, we used the monomeric forms of liver
ferritin, adrenal ferritin, and the 250 kDa protein. Fig-
ure 2B(a) shows that the anti(horse spleen ferritin L
subunit) IgG reacted with all of the samples, revealing
the distinct L subunit band for each sample. When the
same samples were allowed to react with the anti-
(250 kDa protein) Ig, it clearly recognized both the H

and L subunits in liver ferritin, adrenal ferritin and the
250 kDa protein [Fig. 2B(b)], indicating that the anti-
serum raised against the 250 kDa protein is a mixture
of antibodies to the H and L subunits of ferritin.
Because the L subunit was hardly detectable in the adre-
nal cortex ferritin and in the 250 kDa protein, the sam-
ples were overloaded to visualize the L subunit band.
Detection of iron in the 250 kDa protein
To determine whether the 250 kDa protein possesses
the iron storage property of ferritin, we added potas-
sium ferrocyanide to the gel filtration column fractions
[8], which should cause the color of the solution to
turn blue if iron is present. Figure 3A clearly demon-
strates the presence of iron in the 250 kDa protein
peak fractions (Fractions 11–14). Iron was not detec-
ted in any of the fractions that lacked the 250 kDa
protein, eliminating the chance that the detected iron
was an artifact of the purification procedure. The
Fig. 2. Electrophoretic patterns and immunocrossreactivity of the
250 kDa protein and ferritin. (A) Electrophoretic mobility of the
250 kDa protein and ferritin: SDS ⁄ PAGE was carried out with or
without heat treatment of the samples prior to loading in the pres-
ence of SDS detergent, for the monomeric (b) and polymeric (a)
forms, respectively. Lane 1, bovine liver ferritin; lane 2, bovine
adrenal gland ferritin; lane 3, the 250 kDa protein; and lane M,
molecular mass standards (myosin heavy chain, 220 kDa; myosin
light chain 1, 26 kDa; myosin light chain 2, 18 kDa). (B) Immuno-
crossreactivity: monomeric 250 kDa protein and ferritin were trans-
ferred to a PVDF membrane after SDS ⁄ PAGE, and the blots were
incubated with either an anti-(horse spleen ferritin) IgG (Sigma-

Aldrich Japan K.K.) that recognizes the L subunit of ferritin (a) or an
anti-(250 kDa protein) IgG (b). The bound antibodies were detected
by an incubation with horseradish peroxidase-conjugated anti-(rabbit
IgG) IgG (Sigma-Aldrich Japan K.K.). The subsequent staining proce-
dures are described in Experimental procedures. Lanes 1 and 4,
bovine liver ferritin; lanes 2 and 5, bovine adrenal gland ferritin; and
lanes 3 and 6, the 250 kDa protein.
Identification of a putative MAP as ferritin M. R. Hasan et al.
824 FEBS Journal 272 (2005) 822–831 ª 2005 FEBS
absorbance data shown in the upper panel of Fig. 3A
represent the total protein concentrations of the gel fil-
tration column fractions. The SDS ⁄ PAGE patterns
of the corresponding fractions (Fig. 3A: middle panel)
revealed that the protein peak at 280 nm is different
from the peak concentrations of the 250 kDa protein,
because of the presence of contaminating proteins.
Therefore, as shown in the lower panel of Fig. 3A, the
presence ⁄ absence of iron in the gel filtration column
fractions was compared with the presence ⁄ absence of a
visible band corresponding to the 250 kDa protein in
the SDS ⁄ PAGE.
Comparison of iron uptake by ferritin
and the 250 kDa protein
The iron uptake activity was measured by considering
the results of a previous report: when ferritin was incu-
bated with ferrous iron and molecular oxygen in vitro,
an amber colored product [Fe(III)] was formed that
could be monitored by a change in absorbance at
310 nm [16]. The progression plot in Fig. 3B indicates
that the uptake rates of ferritin and that of the

250 kDa protein were almost the same, and were
higher than both controls. The apparent increase in
A
B
Fig. 3. Iron storage and uptake activity of the 250 kDa protein. (A) Detection of iron in the 250 kDa protein: potassium ferrocyanide was
added to a final concentration of 10 m
M to all of the fractions obtained from the gel filtration column chromatography, which was the final
step of the 250 kDa protein purification procedure [8]. The presence of iron was detected by the appearance of a blue color, and was com-
pared with the electrophoretic patterns and the spectrophotometric observations of all fractions. Upper panel, plot showing the absorbance
of all of the fractions from the gel filtration column chromatography at 280 nm, reflecting the total protein contents. Middle panel,
SDS ⁄ PAGE profile of the gel filtration chromatography fractions. The lanes are aligned to the fraction numbers of the plot, in the upper
panel. Lower panel, + ⁄ ) signs are given to indicate the presence ⁄ absence of the 250 kDa protein (first row) and to indicate the pres-
ence ⁄ absence of iron (second row) in the aligned fractions. An increased number of + signs reflects a higher concentration of the 250 kDa
protein as well as the greater intensity of the blue color in the iron detection assay. (B) Iron uptake activity: proteins (either ferritin or the
250 kDa protein) and ferrous sulfate (Fe
2+
) were mixed in 20 MEM to final concentrations of 1.5 mM and 10 nm, respectively, and the
increase in absorbance was monitored at 310 nm for up to 10 min. The data were compared with the progression curves derived from con-
trol 1 (1.5 m
M ferrous sulfate in 20 MEM only) and control 2 (1.5 mM ferrous sulfate + 10 nM BSA).
M. R. Hasan et al. Identification of a putative MAP as ferritin
FEBS Journal 272 (2005) 822–831 ª 2005 FEBS 825
the absorbance in control 1 is due to the auto-oxida-
tion of ferrous iron upon reaction with molecular oxy-
gen. Control 2, which contained an unrelated protein
(BSA) at the same concentration, was included to
observe the effect of a protein in general on the
absorbance data.
Morphological appearances of ferritin and the
250 kDa protein by electron microscopy

Previously, the 250 kDa protein was reported to
appear as a hollow sphere with a diameter of about
12 nm, as determined by electron microscopic observa-
tions [8]. To compare the molecular dimensions of
both bovine adrenal ferritin and the 250 kDa protein,
we observed the negatively stained samples by higher
resolution microscopy, operating at 200 kV. Both of
the proteins appeared to be the same, with an external
diameter of 13 nm and an internal diameter of about
6–7 nm (Fig. 4: A, ferritin; B, 250 kDa protein). The
dark region in the center of each molecule might repre-
sent the iron cores of the ferritin molecules.
Interaction of ferritin with microtubules
To identify whether ferritin binds to microtubules, as
reported for the 250 kDa protein, we examined the
binding of ferritin with taxol-stabilized microtubules
by an in vitro microtubule-binding assay. When tubulin
was excluded from the reaction mixture, the ferritin
remained in the supernatant fraction, but in the pres-
ence of tubulin, a significant portion of the ferritin
sedimented with the microtubule pellet (Fig. 5: lanes 3
and 1). We also found by immunoblotting experiments
that ferritin was present in the mammalian brain
microtubule protein fractions (data not shown). To
clarify the ferritin–microtubule interaction further and
Fig. 4. Electron micrographs of negatively
stained 250 kDa protein and ferritin. Purified
ferritin (A) and the 250 kDa protein (B) were
fixed by 2.5% glutaraldehyde on carbon coa-
ted grids and negatively stained with 2%

uranyl acetate before observation.
Fig. 5. Microtubule binding activity of ferritin. Horse spleen ferritin
(Sigma-Aldrich Japan K.K.) (5 l
M) and tubulin (15 lM) were mixed in
RB containing 30 l
M taxol and 0.5 mM GTP, incubated at 37 °C for
30 min and centrifuged at 16 000 g for 30 min. The resultant
supernatant and pellet were analyzed by SDS ⁄ PAGE (lane 1). Two
control experiments included preparations without ferritin (lane 2)
or without tubulin (lane 3), respectively. s, Supernatant; p, pellet.
Identification of a putative MAP as ferritin M. R. Hasan et al.
826 FEBS Journal 272 (2005) 822–831 ª 2005 FEBS
to demonstrate the binding architecture of ferritin on
microtubules, we made direct observations by electron
microscopy. To exclude the possibility that, on the
electron micrographs, the ferritin might appear in
association with microtubules by chance, we collected
the ferritin–microtubule complex by centrifugation and
redissolved the pellet, while preparing the samples for
electron microscopy. Figure 6 shows the various types
of interactions between ferritin and microtubules.
Ferritin was found to interact randomly with micro-
tubules, either single or in the form of large inter-
molecular aggregates (Fig. 6A–I). Therefore, both the
sedimentation data and the electron microscopy results
clearly demonstrate an association between ferritin and
microtubules in vitro. To investigate whether the iron
within ferritin plays any role in mediating the ferritin–
microtubule interaction, we determined the microtubule
binding ability of apoferritin, the protein shell of fer-

ritin that lacks iron, by an in vitro microtubule-binding
assay. A portion of the apoferritin sedimented with
the microtubule pellet, similar to the iron-containing
ferritin (data not shown), suggesting that the ferritin–
microtubule interaction occurs independently of iron.
We then considered the possibility that the tubulin
preparation, used for the in vitro microtubule binding
assay, might contain trace amounts of residual MAPs
or other noncytoskeletal proteins, and the ferritin
might have interacted with the microtubules, indirectly,
through one or more of these proteins. Therefore, we
observed the effect of total MAP fractions on the ferr-
itin–microtubule interaction. The total MAP fraction
was prepared so that it contained most of the proteins
of the microtubule protein fraction, other than tubulin.
We observed that the addition of increasing concentra-
tions of the total MAP preparation to the reaction
mixtures, containing the same concentrations of tubu-
lin and ferritin, caused a reduction in the amount of
ferritin in the microtubule pellet (Fig. 7: lanes 2–4).
Similar results were obtained, when heat-treated MAPs
(predominantly MAP2 and Tau) were added to the
ferritin ⁄ tubulin reaction mixtures, instead of the total
MAPs (data not shown). Furthermore, Katsuki et al.
[8] showed that the addition of an excess amount of a
MAP4 fragment, containing the microtubule-binding
region, prevented ferritin from binding with micro-
tubules. Altogether, these observations suggest that
ferritin directly interacts with microtubules, and MAPs
or other microtubule associated noncytoskeletal pro-

teins seem to inhibit, rather than facilitate, the ferritin–
microtubule interaction.
Discussion
In this paper, we have described our detailed charac-
terization of a protein that we reported previously on
as a putative MAP, with a relative molecular mass of
250 kDa, which binds microtubules both in vitro and
in vivo. Determination of the primary structures of cer-
tain regions of the protein gave us the first clue that
this protein and the iron storage protein ferritin might
be the same (Fig. 1). Although the absence of the L
subunit sequence and the discrepancy in the molecular
Fig. 6. Electron micrographs showing ferritin–microtubule inter-
action. Ferritin was added to a microtubule preparation reassem-
bled in vitro and the mixture was observed by electron microscopy.
Samples were fixed using 2.5% glutaraldehyde on carbon-coated
grids and negatively stained with 2% uranyl acetate. Ferritin mole-
cules appeared singly (short arrows) or in the form of intermole-
cular aggregates (long arrows) on the microtubule surface.
M. R. Hasan et al. Identification of a putative MAP as ferritin
FEBS Journal 272 (2005) 822–831 ª 2005 FEBS 827
masses suggested that the 250 kDa protein could be a
degradation product or a premature form of ferritin,
further investigations on the molecular masses of the
proteins made it clear that both have the same elec-
trophoretic mobility on SDS ⁄ polyacrylamide gels.
Moreover, the 250 kDa protein was found to dissoci-
ate into two subunits that were indistinguishable from
the ferritin H and L subunits, in terms of their elec-
trophoretic mobility. Although the subunit content of

the 250 kDa protein differed from that of bovine liver
ferritin, it resembled that of adrenal cortex ferritin.
These observations rule out the possibilities that the
250 kDa protein is a degradation product or a precur-
sor form of ferritin. Subsequently, we showed that
antibodies against ferritin and the 250 kDa protein
crossreact with each other, and that the 250 kDa
protein conserves the iron storage ⁄ incorporation
properties of ferritin. Finally, electron microscopic
observations revealed the identical morphological
appearance of both proteins, leaving us with no doubt
that ferritin and the 250 kDa protein are structurally
and functionally the same protein.
The interaction of the 250 kDa protein with micro-
tubules was first described by Katsuki et al. [8].
Although the protein lacked the ability to polymerize
tubulin into microtubules and was structurally distinct
from the other common MAPs, the microtubule bind-
ing characteristics of the protein, as well as the salt
sensitivity and the competition with other MAPs for
microtubule binding, led the authors to conclude that
the protein is a distinct MAP subspecies. However, the
in-depth analysis of the protein, in this study, identi-
fied it as the ubiquitous iron homeostatic protein fer-
ritin, suggesting that the protein should no longer be
considered as belonging to the group of MAPs. Never-
theless, a very important outcome of characterizing the
putative 250 kDa MAP as ferritin is that a novel inter-
action between two very important components of the
cell, namely, microtubules and ferritin, has now been

revealed. The previous report of the binding of the
putative MAP [8], which we have now identified as
‘ferritin’, to microtubules (both in vitro and in vivo)is
further supported by our in vitro sedimentation data,
and our direct observation of ferritin in association
with microtubules by electron microscopy. Moreover,
we found that the addition of a mixture of MAPs and
other microtubule-associated, noncytoskeletal proteins
decreased the extent of the association between ferritin
and microtubules, precluding any chance that the ferri-
tin–microtubule interaction is mediated by another
protein within the microtubule protein fraction.
Subsequently, we noted that apoferritin, which lacks
iron, cosediments with microtubules in a manner sim-
ilar to that observed for the iron-containing ferritin,
indicating that the ferritin–microtubule interaction is
not mediated by the iron stored in the ferritin mole-
cules. Therefore, it is conceivable that the protein por-
tion of the ferritin molecule is responsible for this
interaction, which is most likely to be ionic, as the
addition of salt prevented the ferritin from cosediment-
ing with microtubules in vitro [8]. Thus, the neutraliza-
tion of the anionic microtubule surface by MAPs
could account for the observed inhibition of the ferri-
tin–microtubule interaction by MAPs in vitro. The
intermolecular aggregates of ferritin associated with
microtubules, which we observed by electron micros-
copy, correspond well with the microtubule associated
punctuate structures observed by Katsuki et al. [8] in
Madin–Darby bovine kidney (MDBK) and 3Y1 fibro-

blast cells stained with anti-tubulin mAb and anti-
(250 kDa protein) Ig. The formation of intermolecular
aggregates and their association with microtubules
Fig. 7. Effect of total MAPs on the microtubule binding activity of
ferritin. A total MAP preparation (lane 1) was added to a reaction
mixture (15 l
M tubulin and 5 lM ferritin in RB with 30 lM taxol
and 0.5 m
M GTP), to final concentrations of 0 mgÆmL
)1
(lane 2),
2mgÆmL
)1
(lane 3) and 6 mgÆmL
)1
(lane 4), incubated at 37 °C for
30 min and centrifuged at 16 000 g for 30 min. The contents of the
microtubule pellets were analyzed by 7.5% SDS ⁄ PAGE.
Identification of a putative MAP as ferritin M. R. Hasan et al.
828 FEBS Journal 272 (2005) 822–831 ª 2005 FEBS
might be physiologically significant, in terms of the
stabilization of ferritin molecules from degradation
and the prevention of unwanted iron release into the
cell. Unfortunately, the nature of this aggregation is
unclear at present. It might be an intrinsic feature of
ferritin molecules to aggregate on the microtubule
surface. Alternatively, ferritin molecules might form
cross-bridges between each other through interactions
with free tubulin. In support of the latter explanation,
Katsuki et al. [8] also suggested an interaction between

ferritin and free (nonmicrotubular) tubulin. Further
analyses of the properties and mechanisms of the
association between ferritin and microtubules are cur-
rently underway.
Ferritin exists in a variety of cells and tissue types,
and plays central roles in iron metabolism. In addition,
the presence of ferritin in serum was reportedly corre-
lated with the tissue ferritin and body iron stores.
Although some differences were detected between the
serum ferritin and the intracellular ferritin, the serum
ferritin was shown to be tissue-derived through secre-
tion. Under normal circumstances, equilibrium is
maintained between intracellular and extracellular fer-
ritin, but the concentration of ferritin in serum and
other biological fluids may rise, depending on the iron
status of the body, and under various physiological cir-
cumstances [17–19]. In a couple of studies, Ramm
et al. [20,21] showed that the administration of the
microtubule depolymerizing drugs colchicine and vin-
blastine, in normal and iron-loaded rats, inhibited fer-
ritin uptake and significantly increased the release of
endogenous ferritin in both the serum and bile, sug-
gesting that disturbed microtubule function could
account for these results. These findings also agree
with the fluorescent microscopic observations of Kat-
suki et al. [8], who found that, when MDBK and 3Y1
fibroblast cells were treated with the microtubular
inhibitor nocodazole before staining with anti-tubulin
mAb and anti-(250 kDa protein ⁄ ferritin) Ig, the ferri-
tin that was once associated with the microtubule net-

work to some extent disappeared from the cytoplasm
and accumulated towards the periphery of the cells.
Based on these facts, we presume that the interaction
between ferritin and microtubules and its possible rela-
tionship with microtubule dynamics might be import-
ant in the regulation of ferritin release under different
physiological conditions. On the other hand, the
microtubule-related punctate structures that gathered
around the remaining microtubules after nocodazole
treatment [8] might represent the essential intracellular
pool of ferritin, which remained in the cytoplasm by
forming large intermolecular complexes and interacting
with microtubules. Further studies are required to
reveal the detailed in vivo role of ferritin binding with
microtubules.
Experimental procedures
Chemicals and protein preparations
Taxol was a generous gift from N Lomax (Division of Can-
cer Treatment, National Cancer Institute, Bethesda, MD,
USA). Other reagents used in the study were of reagent
grade, unless otherwise mentioned.
The 250 kDa protein was purified from bovine adrenal
cortex, according to Katsuki et al. [8]. Preparation of ferr-
itin from bovine liver and bovine adrenal cortex was carried
out by following the method described by Ishitani et al.
[22]. Tubulin was purified by phosphocellulose column
chromatography, from a twice-cycled porcine brain micro-
tubule protein fraction, as described previously [23,24].
After the collection of tubulin fractions, the column bound
proteins were eluted by 20 MEM buffer [20 mm Mes

pH 6.8, 0.1 mm EGTA, and 0.5 mm MgCl
2
] containing
0.8 m KCl. The peak fractions were combined, concentra-
ted and dialyzed for subsequent use as the total MAP
fraction.
Amino acid sequence analysis
The purified 250 kDa protein was digested with cyanogen
bromide in 70% (v ⁄ v) formic acid for 24 h at room tem-
perature. The digested products were separated by
SDS ⁄ PAGE and transferred to a poly(vinylidene difluoride)
(PVDF) membrane (Millipore, Bedford, MA, USA) in
transfer buffer [100 m m Tris ⁄ HCl, 192 mm glycine, 20%
(v ⁄ v) methanol, 0.05% SDS, pH 8.3], using an electroblot-
ting system (ATTO, Tokyo, Japan) at 2 mAÆcm
)2
for
90 min. The membrane was stained with 0.1% (w ⁄ v) Ponc-
eau-3R. Three distinct bands were selected, and excised for
sequencing. Sequencing was performed by automated
Edman degradation in a PROCISE
TM
protein sequencer
(Applied Biosystems, Foster City, CA, USA).
Immunoblotting
After SDS⁄ PAGE, the proteins were transferred to a
PVDF membrane, as described above, which was blocked
in blocking buffer [10 mm Tris ⁄ HCl, 100 mm NaCl, 0.1%
(v ⁄ v) Tween 20, 5% (w ⁄ v) skimmed milk, pH 7.5] for 1 h
at room temperature. The blot was then incubated over-

night with either an anti-(250 kDa protein) Ig [8] or an
anti-(horse spleen ferritin) IgG (Sigma-Aldrich Japan K.K.,
Tokyo, Japan) in blocking buffer at 4 °C, washed with
wash buffer [10 mm Tris ⁄ HCl, 100 mm NaCl, 0.1% (v ⁄ v)
Tween 20, pH 7.5], and incubated with a horseradish per-
oxidase-conjugated anti-(rabbit IgG) IgG (Sigma-Aldrich
M. R. Hasan et al. Identification of a putative MAP as ferritin
FEBS Journal 272 (2005) 822–831 ª 2005 FEBS 829
Japan K.K.) for 1 h at room temperature. The membrane
was washed, and the bound antibodies were detected by a
staining solution [0.01% (w ⁄ v) O-dianisidine, 0.03% (w ⁄ v)
4-chloro-1-napthol, 0.01% (v ⁄ v) H
2
O
2
,50mm sodium acet-
ate buffer, pH 5.5].
Detection of iron in the 250 kDa protein
The presence of iron in the 250 kDa protein was detected
by adding potassium ferrocyanide, to a final concentration
of 10 mm, to all fractions obtained from the gel filtra-
tion column chromatography, performed as described by
Katsuki et al. [8]. Fractions that turned blue were consid-
ered to be positive for the presence of iron.
Iron uptake assay
Iron uptake reactions were carried out in 20 MEM buffer,
with 1.5 mm Fe
2+
(ferrous sulfate) and 10 nm protein con-
centrations. Iron uptake by the 250 kDa protein and ferritin

was monitored by measuring the increase in absorbance at
310 nm at room temperature in a UV spectrophotometer
(U 2000, Hitachi, Tokyo, Japan) for up to 10 min.
Microtubule-binding assay
The microtubule-binding assay was carried out in 100 lL
reaction mixtures by adding horse spleen ferritin (5 lm;
Sigma-Aldrich Japan K.K.) to tubulin (15 lm) in reassem-
bly buffer (RB: 100 mm Mes, 0.1 mm EGTA, 0.5 mm
MgCl
2
, pH 6.8) containing 30 l m taxol and 0.5 mm GTP.
The mixture was then incubated at 37 °C for 30 min and
was centrifuged at 16 000 g for 30 min. The pellet was
resuspended in the same volume of RB, and both the super-
natant and the pellet were analyzed by electrophoresis on a
10% SDS ⁄ polyacrylamide gel. Control experiments were
performed in the same way, except that either ferritin or
tubulin was excluded from the preparation. The binding of
ferritin with microtubules in the presence of the total MAP
fraction was also assayed under the same conditions, except
that the total MAP fraction was added to the reaction mix-
tures at concentrations of 0, 2 and 6 mgÆmL
)1
. The con-
tents of the microtubule pellets were then analyzed by 7.5%
SDS ⁄ PAGE.
Electron microscopy
Protein samples were mounted on carbon coated grids (JEOL
substrated grids), fixed with 2.5% glutaraldehyde, and negat-
ively stained with 2% uranyl acetate. Microtubule containing

samples were prepared by adding purified tubulin (15 lm)to
RB containing 20 lm taxol and 0.5 mm GTP. The prepar-
ation was incubated at 37 °C for 10 min, and ferritin was
added to a final concentration of 10 nm. The mixture was
further incubated at 37 °C for 10 min, and centrifuged at
12 000 g for 5 min at 37 °C. The pellet was dissolved in the
same buffer and incubated for 10 min at 37 °C before fix-
ation and staining. Observations were made with a Hitachi
EF-2000 electron microscope operating at 200 kV.
Miscellaneous
SDS ⁄ PAGE was carried out as described by Laemmli [25].
Polymeric 250 kDa protein and ferritin were loaded onto
the gel without heat treatment. To obtain the monomeric
forms, samples were heated at 100 °C for 5 min before
loading. Protein concentrations were determined by the
conventional Lowry method [26], using bovine serum albu-
min as the standard.
Acknowledgements
We would like to thank Dr T. Yasunaga for advice
about electron microscopy. We are grateful to T. Koga,
K. Miyoshi and H. Fujita for generous technical assist-
ance. Thanks are also due to Dr B. Guthrie (SKYBAY
Scientific Editing) for reading the manuscript.
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