Eur. J. Biocheni.
164,485 - 506 (1987)
0FEBS 1987
Review
Iron transport and storage
Robert R. CRICHTON and Mireille CHARLOTEAUX-WAUTERS
Unite de Biochimie, Universite Catholique de Louvain, Louvdin-la-Neuve
(Received October 22/December 12, 1986) - EJB 86 1157
In the development of human society, the utilisation of
metals both in decorative art forms and in objects of practical
application passed through several phases: gold and copper,
malleable and easily workable, were successively replaced by
bronze (an alliance of copper and tin) and thereafter by iron.
It is not the object of the present review to consider the iron
age nor to insist on the important contribution of the Celts in
its development, but the importance of iron in our civilisation
is perhaps best summarized by the quotation from Rudyard
Kipling cited by Philip Aisen in his article on physico-chemical
aspects of iron metabolism [l]:
“Gold is,for the mistress - silver,for the maid -
Copper.for the cruftsman cunning at his trade!
‘Good!’ said the Baron, sitting in his hall,
‘But iron’ - Cold Iron - is master qf them all”.
If the Celts were among the first to recognize the practical
value of this element, it is certain that this was directly
connected with its biological abundance in the earth’s crust,
particularly compared with elements such as gold, silver and
copper [2]. Iron is the second most abundant metal, after
aluminium, and the fourth most abundant element in the
earth’s crust. But it is also interesting to remark that in
contrast to the three elements mentioned above, iron is subject
to an extremely important corrosion, such that there remain
relatively few objects from prehistoric times made with this
metal in comparison with those made from gold, silver or
copper!
In fact, iron, element 26 in the periodic table, has two
properties at physiological pH and in aqueous medium which
make it at the same time interesting for biological systems in
terms of catalysis and transport, and also biologically in-
accessible. Iron can exist in aqueous solution in two oxidation
states: Fe2+ and Fe3+. Secondly, the solution chemistry of
iron is essentially dominated by the hydrolysis and poly-
merisation of aqueous Fe(1II) to insoluble and potentially
biologically inaccessible ferric hydroxides and oxyhydroxides.
We do not consider further this problem, which has been dealt
with in detail in numerous review articles [l, 3, 41, but direct
attention to the consequences which result from this situation.
For living organisms to be able to obtain iron from their
environment they have firstly evolved a series of molecules
(essentially derived from catechols or from a series of different
hydroxamic acids) designated as siderophores [5]. These
Correspondence to R. R. Crichton, Unit6 de Biochimie, Universitk
Catholique de Louvain, Place Louis Pasteur 1, B-1348 Louvain-la-
Neuve, Belgium
Dedicated to Professoi- Dr Gerhard Braunitzer on the occasion
of his 65th birthday.
molecules are powerful complexants of Fe(II1) and are
secreted into the extracellular medium by a large number
of unicellular organisms. The ferri-siderophores [the Fe(II1)-
chelate complexes] are then assimilated by the cells via a
receptor specific for the complex and once inside the cell the
iron is released either by reduction of the iron to Fe(l1) (for
which the siderophore has little affinity) or by hydrolysis of
the ferri-siderophore accompanied by release of its iron.
For multicellular organisms, the problem of the transport
of iron to the different cell types of the organism and its
resorption from the dietary sources of iron available to it
(in general such organisms do not possess a system of
siderophores with their appropriate receptors, and must
therefore find the iron that they require in their alimentation)
also demands a solution. As we shall see, this role is assured
by a serum globulin, transferrin, which complexes iron, trans-
ports it in the circulation and, via specific receptors, is taken
up by the cells of the organism. The release of iron is pH-
dependent and the availability of proton pumps within specific
intracellular compartments assures the intracellular release of
iron; the iron-free protein is recycled from the cell and released
into the circulation for reutilisation.
Once the iron is released within the cell, the same problems
that we have evoked above again present their necessary
fidelity to the rules of the solution chemistry of iron: either
the iron which has been liberated from its soluble siderophore
complex will be complexed by an appropriate intracellular
chelator, or else it will follow its inexorable hydrolysis
and polymerisation towards biological inaccessibility. We
encounter here the second development in the evolution of
the capacity of living organisms to assimilate and utilize iron:
the appearance of intracellular iron storage compounds,
which allow the cell to dispose of a pool of bioavailable iron
in a form which is soluble in physiological conditions, and
non-toxic. The archetype of these compounds is the iron
storage protein, ferritin.
A brief parenthesis imposes itself here: the insistence that
such iron storage compounds should be non-toxic. In fact the
potential toxicity of iron, free in solution through complexa-
tion to whatever ligand it may be, manifests itself through the
reaction described in 1935 by Haber and Weiss [6]: this reac-
tion ( 3 ) is the sum of reactions (1) and ( 2 ) , respectively reduc-
tion of Fe(II1) by superoxide ion and the well-known Fenton
reaction :
Fe(II1) + 0, 4 Fe(I1) + 0 2 (11
Fe(I1) + Hz02 4 Fe(1II) + OH- + OH’ (2)
(3)
486
Since the oxidation of ferrous iron by molecular oxygen
can be represented by the sequence of reactions (4) and (5), it
is immediately apparent that if 'free' iron exists within a cell,
it could provoke the production of hydroxyl radicals, with
disastrous consequences for the cell. The system is in fact used
by macrophages in the killing of bacteria:
Fe(I1) + O2 + Fe(II1) + 0 , (4)
Thus the complexation of iron within a cell necessitates
that the iron storage protein must guard its iron in a soluble,
bio-available and non-toxic form. These conditions are
fulfilled by ferritin.
The importance of iron for living organisms is underlined
by its role in a large number of proteins which require its
presence for their activity [7]. Firstly, there are proteins
involved in the reversible binding of oxygen in animals
(haemoglobins and myoglobins), plants (leghaemoglobin)
and in invertebrates (haemerythrin, this latter containing non-
haem iron). There are many enzymes catalysing reactions with
oxygen : mono-oxygenases such as the pteridine-dependent
hydroxylases for the aromatic amino acids and haemoproteins
such as cytochrome P450; dioxygenases, both haem and non-
haem, for example, tryptophan pyrrolase and prolyl hydroxy-
lase; oxidases, and the terminal enzyme of the mitochondria1
respiratory chain, cytochrome c oxidase; reactions with
peroxides (hydroperoxidases, catalase, ferroxidase, lacto-
ferroxidase) with superoxide (microbial superoxide dis-
mutases) and, associated with other metals, with nitrogen
(Mo) and hydrogen (Ni). In the respiratory, photosynthetic
and microsomal electron transport chains we find
cytochromes (of the types a, b, c and d)as well as iron-sulphur
proteins. Other iron-sulphur proteins catalyse oxidation reac-
tions (xanthine oxidase, xanthine dehydrogenase, aldehyde
oxidase and sulphite oxidase) and the Kreb's cycle enzyme
aconitase is also an iron-sulphur protein (of the 4Fe-4S class).
Finally, last but by no means least, there is the iron-containing
ribonucleotide reductase necessary for the transformation of
ribonucleotide diphosphates to their corresponding deoxy
derivatives.
IRON TRANSPORT IN PROKARYOTES
AND LOWER EUKARYOTES
Introduction
As was pointed out in the opening introduction, micro-
organisms have developed a high-affinity system for iron up-
take from their extracellular medium. This consists of three
components: (a) low-molecular-mass soluble, high-affinity
ferric-iron ligands released by the cells and called siderophores
(from the Greek 'iron bearers'); (b) a membrane receptor for
the iron-loaded form of the siderophore (ferri-siderophore)
that transports the ferric chelate across the microbial mem-
brane; (c) an enzymatic system capable of releasing the iron
from the ferri-siderophore within the cell.
Siderophores are probably found in all aerobic and
facultative anaerobic micro-organisms but they have so far
never been reported in strict anaerobes, in lactic acid bacteria
or in higher organisms.
The functions of siderophores are multiple: they act not
only as receptor-dependent, high-affinity iron transport
systems but also as growth or germination factors, as anti-
biotics or as virulence factors [8 - 101. The iron-free molecules
are used as chelators in the treatment of iron overload in
man.
Siderophores
The first siderophore to be isolated was ferrichrome [ll]
from the culture filtrate of the smut fungus Ustilago
sphaerogena [Ill. It was shown to stimulate the growth of
microorganisms requiring chelated iron. Subsequently the
group of Keller-Scherlein et al. concluded that most actino-
myces produced iron complexing agents, which they called
ferrioxamines [12]. The term siderophore proposed by
Lankford [13] was subsequently adopted and can be defined
[ 5 ] as 'a low-molecular-mass, virtually ferric-specific ligand,
the biosynthesis of which is carefully regulated by iron and
the function of which is to supply iron to the cell'. At present
about 200 different natural siderophores are known [5].
Classification and structure of siderophores
Although siderophores display a considerable structural
variation, they may in general be classified as either
hy droxamates
or phenolates/catecholates (derivatives of 2,3-dihydroxyben-
zoic acid).
13
Siderophores present an extremely high affinity for ferric
iron with formation constants ranging from for entero-
bactin to loz3 for aerobactin [13- 191. Ferric siderophores
are octahedral complexes in which the coordinated metal ion
is d5 high spin and rapidly exchangeable. Since the ligands
are hard bases they have relatively low affinity for soft acid
cations such as Fe(I1) [5]. Thus we could anticipate that iron
release from ferri-siderophores would involve a reduction
step.
The structures of five siderophores, which can all be taken
up as the corresponding ferric complex by Escherichia coli
strain K12, are given in Figs 1- 5 [5].
Ferrichrome, the prototype of hydroxamate-type
siderophores (Fig. 1A) is a cyclic hexapeptide consisting of
three residues of glycine and three residues of N-hydroxy-L-
ornithine in which hydroxamic acid groups are produced by
the acetylation of the three hydroxamine functions (Fig. 1B).
The natural ferrichrome which is produced by many types of
fungi, has a preferred A geometry at the metal centre. Both
Neurospora crassa and E. coli can take up this siderophore (in
the case of the latter organism this despite the fact that it
cannot synthesise it). Moreover, the mirror image A cis-
enantio ferrichrome is ineffective in supplying iron to the fungi
N. crassa [20] and Penicillium parvum [21] and less effective
than the natural A cis ferrichrome in supplying iron to E. coli
P11.

Fig. 1 . Structures of ( A ) ferrichrome, the prototype of the hydroxamate type of siderophores and ( B ) dejerrichrome. (A) Reproduced with
permission from Neilands [5]. (B) * = iron chelation sites. Reproduced with permission from Byers and Arceneaux [336]
A
B
B H j C H , O H
H
C
Fig. 2. Structure of rhodotorulic acid and its derivatives. (A) Rhodo-
torulic acid; (B) dimerumic acid; (C) coprogen. Reproduced with
permission from Neilands [5]
Rhodotorulic acid (Fig. 2) is a dipeptide of acetylated
N-hydroxyornithine which mediates iron uptake in the yeast
Rhodotorula pilimanea and is produced in vast quantities by
the yeast when grown in iron-deficient culture media. This
dihydroxamate siderophore forms a 3 :2 complex with ferric
487
HO -‘C-CO,H
/
R n
- -
schizokinen H 2
aerobactin COOH 4
arthrobartin H 4
Fig. 3. Structure of siderophores based on citric acid. Reproduced with
permission from Neilands [5]
and chromic ions. A recent study has established the
stereospecificity of rhodotorulate-mediated iron uptake in R .
pilimanea [22].
Aerobactin is a derivative of citric acid (Fig. 3) in which
the distal carboxyl groups have been substituted with
hydroxamate functions. In E. coli only strains with the Col V
plasmid, which carries genes for the synthesis of the
siderophore and one gene for the uptake (receptor) system,
can use ferric aerobactin as a service of iron. A number of
other bacteria such as Aerobricter aerogenes can also produce
this siderophore.
The ferrioxamine family of siderophores is represented
(Fig. 4) by desferrioxamine B, the mesylate salt of which is
marketed by Ciba-Geigy as Desferal, the major drug used in
the chelation therapy of secondary iron overload in man.
These siderophores, whch include the antibiotic ferriomycin,
are produced by most actinomycetes [12].
The phenol/catechol type of siderophore enterochelin, or
enterobactin as it is often called, (Fig. 5) is common to all
enteri bacteria. It forms a hexadentate complex with a forma-
488

Fig. 4. Structure of ferrioxumine fumily qf siderophores. Desferrioxamine 9, R = H. Mesylate salt = Desferal. Reproduced with permission
from Neilands [337]

9 A
B

Fig. 5. Structure of ( A ) ferric enterobactin (jerric complex of cyclo-tri-2,3-dihydroxyhenzoylserine) and ( B ) enterohactin (deferri) or
enterochelin. (A) Reproduced with permission from Neilands [5]. (B) * = iron chelation sites. Reproduced with permission from Bycrs and
Arceneaux [336]

tion constant of lo5’ between a ferric iron atom and six Outside Peripi. Cytoplasm
OM CM
deprotonated phenolic hydroxy groups derived from a triester
of 2,3-dihydroxybenzoylserine.The natural iron complex of
enterochelin has the A configuration; the synthetic mirror
image ferric enantioenterochelin does not support the growth
of E. coli [23]. Fez+
We could also add to this collection ferric citrate, for which
a well characterized outer membrane receptor exists [24] in
E. coli.
Fe”

RECEPTORS FOR SIDEROPHORES

Ferric siderophore transport has been extensively studied
in a number of gram-negative bacteria (E. coli, Salmonella
typhimurium), gram-positive bacteria (Bacillus sp. and Myco-
bacterium sp) and in fungi ( N . crassa and U . sphaerogena) [25,
261. However, on account of the well established genetics of
E. coli strain K12, we know most about iron-siderophore
transport in this organism.
At this point it is important to recall that gram-negative
bacteria have both an outer membrane and a plasma (or
cytosolic) membrane separated by a rigid murein (peptido-
glycan) layer. In the outer membrane are located porins, pro-
teins which form pores through which small molecules can
pass into the periplasm and gain access to the plasma mem-
brane, where they may be taken up by specific transport
systems. The molecular mass of siderophores, and more
importantly of their iron complexes, varies over 500-
1000 Da and is thus too large to allow free diffusion through
the small water-filled pores of the outer membrane [27]. It
Fe3‘-Enterocheiin
FE”
Fig. 6. Ferri-siderophores uptuke systems of E. coli K12 pColv. Re-
produced with permission from Hantke [34]
follows as a logical consequence of this observation that they
require a receptor protein.
When E. coli is placed in low-iron growth conditions,
the derepression of seven new outer membrane proteins of
molecular mass 74- 83 kDa is observed [28]. For five of these
proteins (Fig. 6) a receptor function for one, or for several
siderophores has been established: it is interesting to note that
some of these ferri-siderophore receptors also recognise a

number of colicines and bacteriophages. We shall consider
each of these ferri-siderophore receptor systems in turn.
The receptor for ferrichrome, designated in Fig. 6 as fhuA
(ferric hydroxamate uptake) was originally called tonA, the
attachment site for bacteriophages T1, T5 and 6 80 as well as
for colicine and albomycin 1251. The discovery of tonA as
the receptor for ferrichrome in E. coli was preceded by the
discovery of a series of mutants in S . typhimurium resistant to
albomycin, an antibiotic analogue of ferrichrome [28]. The
mutants were found to be defective in ferrichrome transport
and their mapping on the chromosome of S . typhimurium was
found to coincide with the tonA locus in E. coli. As Nielands
has pointed out 1251, this was the origin of the idea that the
biochemical function of the ronA gene product must be that
of a receptor for ferrichrome [29]. While E. coli does not
synthesise ferrichrome, it one the less has an outer membrane
receptor for this ferri-siderophore produced by Penicillium
and many other fungi.
Subsequent to the discovery that the ferrichrome receptor
was the gene product of tonA, it became apparent that the
protective effect of ferric enterochelin against colicine B [30]
could be simply explained by the fact that the colicine B
outer membrane receptor was in reality the ferric enterochelin
receptor. This outer membrane protein of molecular mass
81 kDa, designated in Fig. 6 as fepA, is the receptor for
enterochelin, the siderophore produced by E. coli under low
iron stress.
Strains of E. coli which possess the Col V plasmid can also
synthesise a second siderophore, aerobactin. The receptor
protein for ferric aerobactin, iutA (Fig. 6), present in the outer
membrane, has recently been sequenced [311.
Iron can also be assimilated by E. coli from other
hydroxamate siderophores such as coprogen (from N . crasso),
rhodotorulic acid (from R . pilimanea) and ferrioxamine B
(from various species of Streptomyces), via the fhuE protein
[321.
Finally, E. coli can take up iron from ferric citrate through
the acceptor protein fecA, inducible under low iron stress in
the presence of citrate.
Iron uptake from ferrichromes requires, in addition to
fhuA, at least five other genes: tonB,,fhuCDBand exbB. Three
of them,JhuCDB, map close tofhuA [33], and the gene prod-
ucts offiuC and fhuD are localised in the plasma membrane.
The gene product of tonB is essential for all the ferri-
siderophore uptake systems: mutations in tonB not only
abolish ferrichrome uptake, but all the other ferri-siderophore
uptake systems and that for vitamin B12 are defective. The
same is true for mutations in exbB [34]. Thus, in the
hydroxamate siderophore pathway the specificity resides in
the outer membrane receptor, while the componentsFuCDB,
tonB and exbB are common to all three classes of
hydroxamates (Fig. 6). Ferric enterobactin and ferric citrate
uptake also requires tonB and exbB together with the receptor
protein genes fepA and,fecA and the gene products of,fepB
andfecB respectively (Fig. 6).
IRON RELEASE FROM FERRISIDEROPHORES
Once the ferri-siderophore has been taken up by the outer
membrane receptor protein, iron release can take place by
three mechanisms which are briefly summarised below. It is
somewhat surprising that, whereas iron siderophore uptake
pathways are well established in E. coli, much of our informa-
tion concerning iron release comes from other microorgan-
isms, notably fungi.
489
In the first mechanism, a siderophore may be compared
with an ‘iron taxi’: the ferri-siderophore brings the iron to the
cell membrane but does not penetrate into the cell. Dissocia-
tion of the iron-chelate complex occurs in the membrane: iron
enters the cell while the ligand remains extracellular [17, 35-
371. This mechanism is described in plants and yeast.
The second mechanism proposes the transport of the in-
tact ferri-siderophore into the cell where cellular dissociation
of the metal involves chemical breakdown of the ligand itself.
An esterase specific for the ferric complex hydrolyses the
ligand during iron release in the case of ferric enterobactin
and fusarinine-type siderophores [38 - 421.
This mechanism of iron release from ferri-enterochelin is
typified in E. coli where an esterase, encoded by the gene,f;s,
degrades the enterochelin by hydrolysis of the ester bonds in
the siderophore, and permits iron release with concomitant
destruction of the siderophore [43, 441.
In a third mechanism, the ferri-siderophore is also trans-
ported intact into the cell but the iron is released via a reduc-
tion step. This reaction probably requires a reductase
[NAD(P)H oxidoreductase] which reduces the ferric iron to
the ferrous state; since the siderophore has little affinity for
Fez+ the consequence is that the iron is released. Such re-
ductase activity was reported elsewhere [36, 45 -471. During
this reduction, the ligand may or may not undergo chemical
modification. Enterochelin and ferrichrome were acetylated
during iron release (during or after reduction of iron) and
thus secreted into the external medium [48]. This modified
product had a decreased affinity for ferric iron and so did not
serve as an effective iron siderophore.
In contrast, aerobactin is not destroyed after delivery of
iron: the iron-free siderophore is once again excreted into the
medium and reused in subsequent iron transport [49]. This
mechanism is known as the ‘iron shuttle mechanism’ because
one siderophore molecule compared, as a true ferric iono-
phore, can shuttle more than one iron ion into the organism.
As we will see in the next section of our review, this re-
sembles in many respects the mode of iron transport by trans-
ferrin.
PROTEINS OF IRON TRANSPORT
The purification of iron binding proteins in extracellular
biological fluids has a long history. Conalbumin (which we
now refer to as ovotransferrin) was purified in 1900 [50] and
identified as the antimicrobial agent of raw egg white in 1946
[51]. Two years earlier it had been established that the anti-
microbial properties of egg white were abolished by the addi-
tion of iron [52]. The iron binding protein of human serum,
serotransferrin was purified by Schade and Caroline in 1946
[53] and its anti-bacterial properties, as well as their abolition
by iron, established. Working independently, Laurell and In-
gelman [54] also purified the ‘red’ protein from pig plasma
and in the same year Holmberg and Laurell [55] proposed
the name ‘transferrin’, which has since replaced the name
siderophilin originally utilised by the American workers. In
1949 Schade et al. [56] established that human serotransferrin
binds two atoms of iron per molecule and that iron binding
is accompanied by the concomitant binding of a bicarbonate
ion for each iron atom bound. In the same year it was estab-
lished that transferrin iron can be released [57] by acidification
of the medium. Subsequent studies have clearly established
the importance of this mechanism within the cell. The iron
binding protein of milk, lactotransferrin, is a relative new-
comer to the scene [58,59] but its iinportance both as an iron
490
I S , 10 15

LTF
STF
OTF
LTF
STF
OTF

9*
Q P R T H
D P Q T F
G S T T S
bs w w

C S K T D E R P A S

LTF L L F ?o T c s ? K F Dy.-mQ sIA[G sr-p R s

STF LL Y N K I N H C R F D El FFSEGCAPGSK-KDS
OTF L I H NOR T G T c N F D EI Y F S E G C A P G S P- N S

0 6 a

LTF P R I D ? G L!P' S G:fF?A I Q N%!K S E?E V?

Fig. 7. Amino acid sequences of human serotransferrin ( S T F ) , hen ovotransferrin ( O T F ) and human lactotransferrin (LTF). Reproduced with
permission from Metz-Boutigue et al. [73]

NeuAc(aZ-b)-Gal(R1-4)-GlcNAc(~1-2)-Wan(al-3)
A Man( B1-4)-GlcNAc(B1-4)-G1 cNAc t l - A s n
NeuAc(crZ-6)-Gal(B1-4)-Cl cNAc(Rl-Z)-Man(a1-6)
NeuAc(x2-6)-Gal(B1-4)-GlcNAc(B1-2)-Man(nl-3)
B \Man
NeuAc(a2-6)-Gal(~l-4)-GlcNAc(B1-2)-Man(~l-6)
GlcNAc( P I - 4 )
\
GlcNAc(~1-2)-Man(a1-3)
C
G1 cNAc( B1-4)
GlcNAc( p l - E ) - Y a n ( B 1 - 6 )
Fig. 8. Structure of the principal glycans of ( A ) human serotransferrin, ( B ) human lactotransferrin and ( C ) hen ovotransferrin. Reproduced
with permission from Spik et al. [338]
transport protein and as a protective agent against bacterial
infections (it is found also in tears and is secreted by
leukocytes) is now well established. As we shall see, the ex-
tensive sequence homologies between the three types of trans-
ferrin, not only confirm the presence of two distinct domains
but allow us to draw some conclusions concerning the nature
and localisation of their iron binding sites.
Structure of trandferrins
The transferrins of all of the three classes described above
are glycoproteins of molecular mass around 80 kDa which
have the capacity to bind two atoms of ferric iron in associ-
ation with the binding of an anion, usually bicarbonate (for
reviews see [60, 611). The form of the transferrin molecule, as
deduced from preliminary X-ray crystallographic studies of
human and rabbit serotransferrin [62 - 641 and human
lactoferrin [65] and from hydrodynamic data [66], is that of
an ellipsoid of revolution with a ratio of 2 : l between the
major and minor axes. The rabbit serotransferrin molecule
presents a clearly dilobal structure [64] but it is at present
premature to suggest that the two lobes represent the two
distinct N- and C-terminal iron binding domains to which we
will refer subsequently.
The amino acid sequences of human serotransferrin [67 -
701, of hen ovotransferrin [71, 721 and of human lacto-
transferrin [73] have been determined and are presented in
Fig. 7. The sequence homology between human sero-
transferrin and human lactotransferrin or hen ovotransferrin
is 59% and 51 7'0 and between human lactotransferrin and hen
ovotransferrin is 49%. The homology is more pronounced in
the C-terminal half of the molecule in all three cases. When
the N-terminal and C-terminal halves of the three transferrin
molecules are compared there is clear evidence of internal
homology: 41 '/O for human serotransferrin, 37% for human
lactotransferrin and 33% for hen ovotransferrin. For human
lactoferrin [73] clear evidence was found for a sequence hom-
ology of the N- and C-terminal halves (1 - 338; 339 - 703) by
the method of Staden [74], but no evidence was found for the
fourfold or sixfold internal homology previously reported [71,
75, 761. However, results obtained using the method of Smith
49 1
/
(?1-4)-G1 cAC( Bl-4)-G1 cNAc P 1- Asn
/ (31-6)
I
Fuc
1 Man( p l - 4 ) - G 1 cNAc( 41-4) -G1 cNAc l11-Asn
/
and Waterman [77] strongly suggest the presence of two hom-
ologous domains (44% identical residues): ND 2 (108 -232)
and CD 2 (463 - 589), which could be taken as an indication
that each half of the molecule is composed of three distinct
domains. For the moment, the two-domain nature of trans-
ferrins seems to be well established and, as we will see, each
of these domains contains an iron binding site.
T h e carbohydrate component of transferrins
The transferrins are glycoproteins. Human serotransferrin
contains two branched oligosaccharide chains attached to the
asparagine residues 415 and 608. The structure of the glycan,
determined by chemical and enzymatic techniques and con-
firmed by NMR and mass spectrometry [78 - 801 is given in
Fig. 8. The glycosylation sites conform to the rule that the
sequence which codes for glycosylation is the tripeptide Asn-
Xaa-(Ser/Thr), namely Asn-Lys-Ser (residues 428 -430) and '
Asn-Val-Thr (635 - 637)'. Both are in the C-terminal half of
the molecule, and no other potential glycosylation sites are
apparent in the amino acid sequence [67-701. In human
lactotransferrin [73] there are four potential sites for glycosyla-
tion: Asn-Trp-Thr (137- 139), Asn-Ala-Ser (389-393), Asn-
Gln-Thr (490 - 492) and Asn-Gly-Ser (635 - 637). Only two
of them are glycosylated [73], namely the asparagine residues
at positions 137 and 490 of the lactoferrin sequence. Hen
ovotransferrin contains three sequences in the C-terminal half
of the molecule respectively Asn-Arg-Thr (490 -492) ', Asn-
Gly-Ser (635 -637)' and Asn-Pro-Ser (690-692)', which
could serve as sites for glycosylation. Only one of them
appears to be glycosylated, namely asparagine 490. We might
conclude from this that the presence of a residue Xaa which
has a sufficiently small side chain, and thus permits flexibility
of the polypeptide chain (Ala or Gly) or an amino acid which
imposes rigidity to the main chain (Pro) blocks the action of
the glycosylation in Asn.
' The numbering of rcsidues in human serotransferrin and hen
ovotransferrin are based on the sequence of human lactotransferrin
(Fig. 7).
492
1
I 4&1
Fig. 9. Localisation of the disulfide bridges of human lactotransferrin ( L F T ) , human serotransferrin (STF) , and hen ovotransferrin (OTFj.
Reproduced with permission from Metz-Boutigue et al. [73]
The biantennary glycan given in Fig. 8 is found in both
glycosylation sites A and B of human serotransferrin. Howev-
er, in addition, other structures, notably triantennary glycans,
are also found in normal human serum transferrin [81, 821
albeit in much smaller amounts. The structures of the human
lactoferrin glycans [83] and of the ovotransferrin glycan [84]
are also given in Fig. 8. While the core glycan remains the
same as for serotransferrin, there are quite substantial
differences in the degree of substitution, notably fucosylation
of GlcNAc residues in lactotransferrin, and introduction of
GlcNAc on mannose residues in ovotransferrin.
Disulphide localisation in transferrins
The distribution of half-cystine residues and, for that
matter, of disulphide bridges in serotransferrin, lactotrans-
ferrin and ovotransferrin is remarkably well conserved
(Fig. 9). Thus, in the N-terminal half of the molecule, six
disulphide bridges (numbered 1 - 6 according to Williams
[SS]) are found in essentially similar positions in all three
molecules. The same is also true of nine disulphide bridges in
the C-terminal half of the molecule (for the clarity of the
discussion, we ignore a number of additional disulphides pres-
ent in one or other of the transferrins). Of these nine di-
sulphides, six are in more or less homologous positions with
those of the N-terminal half of the molecule. On the basis of
these data, Williams has proposed [85]that the precursor of
transferrin as we know it today was a molecule which re-
sembled the N-terminal half of the molecule with a molecular
mass of 40 kDa, one iron binding site and six disulphide
bridges. To explain the appearance of the product of gene
duplication that we find in the present day transferrins, he
suggests that the 40-kDa protein was too small to avoid elimi-
nation from the circulation by the kidneys, and cites results
with N- and C-terminal domains of ovotransferrin 1861which
confirm this hypothesis.
In conclusion, the conservative dispositon of the di-
sulphide bridges in transferrins strongly supports the idea
that the transferrins are the product of gene duplication and
reinforces the two-domain nature of these proteins.
The iron and anion binding sites of transferrins
The binding of Fe3+ to each of the two binding sites in
the N- and C-terminal domains of the transferrin molecule
is accompanied by the binding of an anion (carbonate or
bicarbonate) and by the release of three protons. Whether
these latter are derived from hydrolysis of water coordinated
to the iron, from ionisable groups of the protein, or from both
sources remains unclear [60]. The consequence of iron binding
is that the net negative charge of the transferrin molecule is
increased by one unit for each iron bound [87]. These early
studies of iron binding [87] suggested cooperativity between
the sites of ovotransferrin such that the affinity of the second
site for iron was increased by two orders of magnitude
following binding of iron to the first site. However, subsequent
studies on human serotransferrin indicated that iron binding
was equivalent for the two sites [88-901. A number of
spectroscopic studies (reviewed in 1601) however revealed
differences between the two metal binding sites of ovo-
transferrin. This view was substantiated by results which
showed that the one of the two iron binding sites of human
serotransferrin was much more susceptible to release by pro-
tons than the other [91, 921, and by the demonstration [93]
that the N- and C-terminal domains of ovotransferrin are
occupied by iron in a clearly non-equivalent manner. A
detailed analysis was undertaken by Aisen, taking advantage
of the method developed by Makey and Seal [94] which, for
the first time, enabled the four distinct species of transferrin,
namely apotransferrin, monoferric transferrin A, monoferric
transferrin B and diferric transferrin, to be separated one from
the other on polyacrylamide gels in the presence of urea.
Whereas at pH 7.4 the affinity of the C-terminal site is 5 -6
times greater than that of the N-terminal site, the difference
at pH 6.7 is 20 times [95]. The affinity of the two sites in
circulating serotransferrin under physiological conditions
ranges over 1-6 x 10” M-’ [961.
The identification of the amino acid residues involved in
the iron and anion binding sites must ultimately await the
determination of the three-dimensional structure from X-ray
crystallographic studies. However, in view of the fact that the
EPR and optical spectra of different transferrins are very

similar [88, 971 and that the amino acid residues which
constitute these sites would be expected to be conserved in the
course of evolution, it is not unreasonable to attempt at pres-
ent to identify them. On the basis of results obtained by a
number of techniques, it seems likely that the iron ligands in
transferrins involve two histidine residues [98, 991, two or
three tyrosine residues [loo, 1011, a water molecule or a
hydroxide ion [loo, 1021, and a carbonate or bicarbonate
anion [loo, 1031. This latter is bound not only to the metal
but also to the protein: chemical modification studies [lo41
imply that an arginine is involved, whereas NMR
spectroscopy suggests that histidine is the anion binding
ligand of the protein [loll.
A tentative analysis of the iron and anion binding sites of
transferrins was made by Chasteen [lo51 and in the present
analysis we have included the sequence data of the human
lactotransferrin molecule [65].
The conclusions of Chasteen are in part supported and
clarified by the inclusion of the human lactoferrin sequence
(Fig. 7). For the two histidine ligands it seems likely that they
are 117 and 252 for the N-domain and 472 and 609 for the C-
domain (all three proteins have histidines at these positions)
and that the histidine residues at positions 210 and 558 are
not ligands since in human lactoferrin they are replaced by
glutamate and aspartate respectively. The assigment of
tyrosine ligands proposed by Chasteen (188 and 191 for the
N-domain, 538 and 540 for the C-domain) is compromised
by the presence of a lysine residue at position 188 of the
lactoferrin sequence, and also by the presence of tyrosine
residues in all three transferrin sequences for both N- and C-
terminal domains at positions 83 and 427, and at positions 93
and 447. Since the tyrosine residues at positions 93, 188 and
191 in ovotransferrin are protected by iron from reaction with
tetranitromethane [loll, we might speculate that perhaps the
two tyrosine ligands are those at positions 93 and 191 for the
N-domain, and those at positions 447 and 540 for the C-
domain.
For the arginine residues involved in the binding of the
anion, Chasteen opts for the residues at positions 121 and 477
in the N- and C-domains, which are effectively conserved in all
three transferrin sequences. The involvement of the arginine
residues at positions 235 and 257 in the N-domain, and at
positions 592 and 614 in the C-domain, which Chasteen re-
cognises cannot be excluded and which are conserved in all
three transferrin molecules, might be eliminated by the prox-
imity of conserved histidine residues at positions 117 and 472,
thereby accounting for the perturbation of the proton NMR
spectra of histidine residues concomitant on iron and anion
binding [99].
Transferrin iron uptake and release
The form in which iron is presented to the apotransferrin
molecule in vivo is unknown. From in vitro studies it appears
that Fe(I1) and Fe(II1) can serve as a source of transferrin
iron. The former is autoxidised by the protein through a
chelate effect [106]; Fe(I1) is poorly bound by the protein, if
at all [107], whereas Fe(II1) is very tightly bound. When simple
ferric salts are used they hydrolyse, polymerise to complex
polynuclears and result in non-specific binding [log]. This can
be avoided by the use of ferric chelates or freshly prepared
solutions of ferrous salts [109]. An excess of HCO; in solution
ensures the occupation of the anion binding site by the physio-
logical anion, and furthermore ensures specific binding [109].
In the case of ferric nitrilotriacetate, which is often employed
493
for the preparation of 59Fe-labelled transferrins [110, 1111,
iron binding appears to involve a two-step process. First a
spectroscopically distinct and identifiable ternary complex of
ferric nitrilotriacetate and the protein is formed, in which
the anion binding site is occupied by nitrilotriacetate [112].
Thereafter, in a much slower reaction, the bicarbonate ion
replaces nitrilotriacetate at the anion binding site to give the
physiological form of transferrin. While this model of iron
binding may be true for ferric nitrilotriacetate, it is by no
means sure that it applies to ferric citrate or for that matter
to any other as yet unidentified physiological ferric chelate.
An alternative model for iron binding based on the obser-
vation that bicarbonate and oxalate bind, albeit weakly, to
apotransferrin [113], assumes that anion binding precedes iron
binding [114]. Anion binding neutralises positively charged
groups on the protein and thus facilitates the approach of
Fe(II1) or Fe(1I) (the latter is subsequently autoxidised) to the
binding sites. The protonated groups in the vicinity of the
metal binding site may be the source of some of the protons
which are released when apotransferrin binds iron.
The release of iron from transferrin is most readily
achieved in vitro by lowering the pH to values of between
5.0 - 5.5 in the presence of appropriate Fe(II1) chelators,
although for lactotransferrin much more extreme pH values
of 2 - 3 are required for iron release [115]. It is possible that
protonation of the anion in the metal binding site destabilises
the iron-anion-protein ternary complex and that this leads to
release of the anion and of the metal. As we shall see in the
next part of this review, the physiological consequences of the
inclusion of transferrin within an acidic intracellular vesicle
results de facto in the release of its iron within the cell.
A number of other models have been proposed for trans-
ferrin iron release. One involves direct chelation of the trans-
ferrin iron by stronger iron chelators such as bacterial
siderophores. However, whereas catechol-based siderophores
and synthetic analogues are both thermodynamically and
kinetically capable of removing transferrin iron [116, 1171,
desferrioxamine B although thermodynamically capable, is
kinetically slow both in vivo and in vitro [118,119]. The kinetic
barrier to iron release by desferrioxamine B can be overcome
by a number of iron-chelating anions of which pyrophosphate
and organic pyrophosphates are most effective [120, 1211. The
introduction of a catechol group on the terminal amino group
of desferrioxamine B gave a derivative which was kinetically
able to remove transferrin iron [122]. The cationic groups near
the metal binding site may serve to direct anionic chelating
agents to the iron and participate in recently observed
quaternary complexes of the type chelate-iron-transferrin-
carbonate [123].
Mechanisms involving reducing agents have also been pro-
posed [124, 1251 although their physiological relevance re-
mains unclear (in view of the nature of the reducing agents
employed).
Transferrin iron uptake by cells
In a paper published in 1963, Jandl and Katz [126] pro-
vided convincing evidence that doubly labelled transferrin
delivered its iron to reticulocytes by a process in which the
protein remained to a large extent in the stroma whereas the
iron was internalised within the cells. This plasma to cell
cycle of transferrin could best be explained by a receptor for
transferrin on the plasma membrane of the cel!s which allowed
the iron-loaded transferrin molecule to bind to the cell surface,
release its iron and thereafter the iron-depleted protein mole-
494
cule would be released to renew its search for iron and to
initiate a new cycle of transferrin-mediated iron uptake.
As we will see in what follows, the fundamental principle
of the transferrin cycle has proved to be valid for almost all
cell types which have been examined, and has been clarified
in a great many of its details for an already impressive number
of different cells. However, before discussing in detail the
mechanism by which cells take up transferrin, release its iron
within the cell, and recycle the apotransferrin back to the
circulation, we might well ask the question: how do cells
regulate their requirement for iron? The answer comes from
an elegant study by Morgan and his colleagues [127]. An
analysis of the number of transferrin receptors and the rate of
transferrin-bound iron uptake by various immature erythroid
cell precursors showed that during erythroid cell development
the number of transferrin receptors increased from 300000
per cell at the early normoblast stage to a maximum of 800000
per cell on intermediate normoblasts. The further maturation
of intermediate normoblasts was accompanied by a decline in
the number of transferrin receptors, which attained a level of
105000 per cell in the circulating reticulocyte (and which
declines to almost imperceptible levels in mature red blood
cells). A close correlation was observed between the number
of transferrin receptors and the iron uptake from transferrin
during erythroid cell development, and it was found that in
all of the immature erythroid cell populations studied the rate
of iron uptake was about 36 iron atoms per receptor per hour.
The conclusion of the authors, and their circumspection in
expressing it, merits citation: 'These results indicate that the
level of transferrin receptors may be the major factor which
determines the rate of iron uptake during erythroid cell
development'. We can only add that this conclusion seems
likely to be true for other cell types than those of the erythron,
since we know that many tumour cells express much greater
numbers of transferrin receptors than do the corresponding
normal cells, and that their reaction to a culture medium
which seeks to deprive them of iron is to increase the number
of transferrin receptors at their cell surface severalfold [128].
However, we do not yet know the mechanisms by which a
mammalian cell regulates its expression of transferrin re-
ceptors as a function of its needs for iron.
The transferrin receptor
The discovery of the transferrin receptor resulted from
the preparation of monoclonal antibodies against a human
erythroid leukemia cell line. The monoclonal B3/25 was in
fact found to be directed against the transferrin receptor [129],
as was a monoclonal anti-T cell reagent OKT 9 [130]. Indeed,
it seems likely that many monoclonal antibodies directed
against tumour cell lines are directed against the transferrin
receptor [131]. The structural features of the transferrin re-
ceptor are depicted in Fig. 10 (from [132]).The model is based
on data published in [129, 130, 132-1371. The receptor is a
disulphide-linked dimer of molecular mass 180 kDa which
presents two transferrin binding sites. Trypsin digestion
liberates a 70-kDa fragment whch retains the capacity to bind
transferrin and which is also able to bind the monoclonal
antibodies B3/25 and OKT 9. The receptor has N-asparagine-
linked glycans probably composed of two high-mannose
chains and one complex chain. The antigenic determinants
which react with OKT 9 and B3/25 are part of the polypeptide
and are not the glycans. In the internal part of the receptor
the transmembrane tail of molecular mass 5 kDa possesses
phosphoryl serine residues and a covalently bound fatty acid.
90
I
Fig. 10. The human serotransferrin receptor. Reproduced with
permission from Newinan et al. [132]. Numbers on the right indicate
molecular mass in kDa
The gene for the human transferrin receptor has been mapped
on chromosome 3 [138, 1391 and two cDNA clones for the
receptor have recently been isolated [140]. The gene for trans-
ferrin is also on chromosome 3 [138] as is the gene for the
melanoma cell-surface antigen, p97, which has sequence ho-
mology with transferrin and binds iron [141]. The avian bursa1
lymphoma oncogene (B-lym) codes for a polypeptide that is
structurally related to transferrin, lactoferrin and p97 [142].
The transferrin cell cycle
As we have seen earlier, the idea of a plasma-to-cell cycle
of transferrin was first evoked by Jandl and Katz [126]. The
half-life of human serotransferrin is 7-9 days [143]. When we
take into account that 30-40 mg iron is exchanged between
different cellular compartments in man each day [144], it is
immediately apparent that the transferrin molecule must be
recycled intact many times in the course of delivery of iron to
cells.
Our ideas concerning the serotransferrin cell cycle have
evolved gradually over the past decade since the idea that iron
release from transferrin might take place in a vesicle with a
localised low pH was first mooted [145]. It was assumed from
early studies that transferrin uptake was only dependent on
the interaction with receptors at the cell surface [126], and this
view point continues to receive support as we will see later.
However, the fact that a number of features of the process of
transferrin and iron uptake were similar to those observed in
the endocytosis of other proteins by cells led to the suggestion
that transferrin might enter immature red cells prior to the
release of its iron for haem synthesis [146]. Evidence was
accumulated which showed that subsequent to binding of the
iron-loaded transferrin to its plasma membrane receptor, the
transferrin-receptor complex entered erythroid cells by endo-
cytosis [145- 1501.
What might be the intracellular pathway of the transferrin-
receptor complex which results in release of its iron within the
cell and in the recycling of the apoprotein back to the plasma
membrane? Using antibodies directed against the plasma
membrane of cultured rat fibroblasts it was concluded that

< Receptor
It, Oiferric tronsferrin
0 Apotronsferrin
+ Fe
Fig. 11. Transferrin uptake and recycling in human tumor cell: the
transferrin cell cycle. Reproduced with permission from Dautry-
Varsat et al. [I711
plasma membrane fragments, after internalisation, gain access
to lysosomes and are thereafter recycled back to the cell
surface [151]. By analogy, we proposed that the transferrin-
receptor complex passed into the lysosomal compartment
where its iron was released, and that the apotransferrin mole-
cule was returned to the cell surface and discharged into
the extracellular medium [152]. Further, it was shown that
a number of drugs which affect endocytosis and lysosomal
function greatly reduce iron uptake from transferrin by
fibroblasts without significantly affecting the uptake of trans-
ferrin protein [152 - 1541. Similar results were obtained from
studies on developmentally totipotent teratcarcinoma stem
cells [155] and it was again concluded that the lysosome was
an important intermediate in the iron uptake pathway. In
parallel investigations on reticulocytes, the group of Morgan
found support for the hypothesis that iron release from trans-
ferrin occurred as the result of protonation of transferrin
within acidic intracellular vesicles [156- 1611. Similar effects
of lysosomotropic agents were observed in human leukemic
and hepatoma cell lines [162,163]. That the intracellular acidic
compartment was nonlysosomal in nature was established by
Percoll gradient fractionation of postnuclear supernatants in
K562 human erythroleukemic cells [164]. The intracellular
route of transferrin and its receptor via a 'short circuit' re-
cycling ligand and receptor to the cell surface was found in a
number of tumour cells [165-1691.
The effect of pH on the binding of transferrin and
apotransferrin to its receptor on rabbit reticulocytes and on
the human tumour cell lines HepG2 and K562 was analysed
and led to the formulation of the transferrin-cell cycle which
is illustrated in Fig. 11 [170- 1721. Subsequent to the binding
of the iron-loaded transferrin to its receptor, the complex is
internalised, with formation of a coated pit and a coated
vesicle [173-1751 which sheds its clathrin to become an
endosome. An energy-dependent proton pump acidifies the
pH of the endocytic vesicle to pH 5.5-5.0. Under these
conditions, but probably also with the help of an as yet un-
identified intraendosomal chelator, the iron, bound to the
transferrin-receptor complex, is released and passes into the
49 5
cytoplasm. In contrast to many other ligand-receptor com-
plexes, the apotransferrin-receptor complex retains a high
stability at acidicpH such that after its transition in the CURL
(compartment of uncoupling of ligand and receptor) it is
recycled back to the plasma membrane. At the neutral or
slightly alkaline pH of the extracellular fluid, apotransferrin
has a lowered affinity for its receptor, is released, and can
return to the circulation in search of iron.
That this mechanism occurs in other normal cell types is
supported by studies on erythroid, placental and hepatic cells
[170, 176- 1841. In rat liver most of the transferrin was found
in the hepatocytes with little in other cell types (Kupffer cells,
endothelial cells, fat cells) and the iron was found to be rapidly
incorporated into mitochondria1 and cytosolic ferritin. Evi-
dence was also found for the presence of transferrin within
a low-density membrane fraction [176, 1771. The uptake of
transferrin by reticulocytes occurs by endocytosis through
coated pits [178,179] and the rate of endocytosis of transferrin
is equivalent to that of iron uptake [160]. Further, the inhibi-
tion of iron uptake by inhibitors of endocytosis is pro-
portional to the inhibition of transferrin endocytosis [l SO] and
both transferrin and its receptor are recycled to the cell surface
by exocytosis [181] with a recycling time of 3-4 min at 37°C
[160]. That receptor binding [182] and endocytosis [183] is also
involved in transferrin and iron uptake by placenta is also
well established.
However in placenta and in hepatocytes there seem to be
two intracellular pathways of endocytosed transferrin, one
recycling rapidly (and responsable for the bulk of the iron
uptake) and a second recycling more slowly [184].
As has been noted above, if recent results seem to point
to the role of this transferrin cycle in many cell types, both
normal and tumoral, there are still reports in the literature
which imply that at least part of iron release from transferrin
takes place at the plasma membrane, based on studies with
rat and rabbit reticulocytes [185 - 1921.
PROTEINS OF IRON STORAGF
Introduction
In animals, the major pool of non-haem iron (ap-
proximately 25% of total body iron) is stored in two
compounds: ferritin and haeinosiderin [143]. Ferritin was first
crystallised from horse spleen by Laufberger in 1935 and in a
detailed publication [193] he described its purification, its high
iron content (over 20% by weight) and suggested that it was
probably a substance that served as an iron depot. It has a
widespread distribution in all kinds of living organisms; its
presence has been reported in mammals, birds, fish, in-
vertebrates, plants, fungi and in prokaryotes such as E. coli
and Azotobucter sp (reviewed in [194-1981). Ferritin is also
present in serum, where it was first discovered as the active
component of a vasopressor substance [199]; its determination
in serum is useful in the assessment of iron storage status
[200]. Haemosiderin was first isolated from horse spleen in
1929 as insoluble red-brown granules [201]. Both ferritin and
haemosiderin have a high and variable content of iron: the
former is found in the cytoplasm in a soluble form whereas
the latter is present as insoluble granules within secondary
lysosomes. Both iron storage compounds consist of an in-
organic core, the iron mineral of which has a rather similar
composition and structure, and a protein component, which
is well characterised in the case of ferritin, but remains ill-
defined for haemosiderin.
496
. . . . . . . . . . . . . . . . . . . . .
Fig. 12. Structure of ferrihydride. Reproduced with permission from
Ford et al. [264]
Ferritin structure: the mineral core
As we have noted above, the ferritin molecule consists of
two components: a mineral core of hydrated ferric oxide and
a multi-subunit protein shell which englobes the former and
thereby assures its solubility in an aqueous environment. The
ferritin molecule can maintain a concentration of 1 M ferric
iron in solution, in contrast with the extremely poor solubility
of the variety of sols, gels, amorphous or crystalline precipates
that emerge from the hydrolysis of iron(II1) in aqueous solu-
tion [4].
The mineral core of ferritin is an inorganic hydrated
iron(II1) oxide of variable iron content containing some
phosphate: for the ferritin of horse spleen an atomic ratio,
Fe/P = 9, was reported in early studies [202] whereas a more
recent investigation [203] found ratios of 21 for human spleen
ferritin and of only 1.7 for bacterial ferritin from Pseudomonas
aeruginosa. The iron content of the horse spleen ferritin mole-
cule varies from 0 to a maximum of 4500 atoms [204]. Re-
constitution of ferritin in the absence of phosphate has no
effect on the X-ray or electron diffraction pattern [205], and
treatment of ferritin with 1 M NaOH, which provokes the
dissociation of the protein shell, results in the loss of more
than 80% of the inorganic phosphate originally associated
with the iron core [206]. Analysis of the X-ray and the electron
diffraction patterns of ferritin iron cores 1207-2101 indicate
that the ferritin iron core has a structure which resembles that
of the mineral ferrihydrite [211, 2121; it is illustrated in Fig. 12.
This mineral, (5Fe203 . 9H20), which is formed by the slow
heating of Fe(N03)3 . 9 H 2 0 at 80 - 85 "C [207], has been
found in the walls of old mine workings as a relatively recent
deposit [212]. The unit cell contains four oxygen layers (in-
stead of six in the closely related haematite structure) and the
Fe(II1) sites are underpopulated with a stoichiometry of 5Fe :
120 (instead of 2: 3 in haematite). Alternative structures based
on radial X-ray scattering [213] or on extended X-ray absorp-
tion fine structure (EXAFS) measurements [214, 2151 are ex-
cluded by the absence of octahedral iron and incompatibility
with the X-ray and electron diffraction patterns respectively.
The localization of the phosphate ions in the ferritin cores
remains unknown. In vitro it can be replaced by dissolved
phosphate ions [216, 2171. However, inclusion of phosphate
in the reaction mixture yields a more monodisperse core size
[216] and a smaller apparent size when measured by Moss-
bauer spectroscopy [218]. The phosphate ions might replace
surface hydroxyl functions. This could explain the evidence
from neutron low-angle scattering [219] and electron micros-
copy [220]which are both compatible with a direct interaction
between the core and the protein shell. As we will see this
would also account for the presence of a number of lysine and
arginine residues in the interior of the apoferritin molecule
[215] which could interact with phosphate residues on the
surface of the iron core and help to bind the hydrated iron
oxide to the inner surface of the protein.
Haemosiderin has considerable spectroscopic and mor-
phological similarities to the ferritin iron core [221-2241 de-
spite its much higher ironlprotein ratio.
One hypothesis is that soluble cytosol ferritin is pro-
gressively transferred to and accumulated within scondary
lysosomes: the following sequence of events has been pro-
posed for the formation of haemosiderin [221]:
Cytosol ferritin + lysosome-siderosome ferritin
1
denaturation of ferritin protein
(loss of solubility, aggregation)
5-
proteases
5-
decomposition of ferritin protein
1
divested (FeO . OH) cores
5-
core disintegration
1
amorphous haemosiderin (FeO ' OH).
This would be consistent with data which indicate the
presence in human and horse ferritins of high-molecular-mass
polymers which are less soluble and more susceptible to pre-
cipitation than ferritin monomers and which have an iron/
protein ratio consistent with loss of protein from this ferritin
[225].
Apoferritin: the protein subunit
Treatment of ferritin with reducing agents such as dithio-
nite, together with an appropriate chelator of Fe(I1) [226]
or thioglycollate [ 1941, followed dialysis or gel permeation
chromatography to remove the iron-chelate complex and the
reductant yields the intact protein shell, apoferritin. Native
apoferritin can be prepared by differential or density gradient
centrifugation of ferritin solutions. Both types of apoferritin
are closely similar as judged by X-ray diffraction [204, 2271,
circular dichroism and hydrodynamic properties [228]. Most
structural as well as functional studies have been carried out
with chemically prepared apoferritin. Horse spleen apoferritin
has a molecular mass of 476 kDa and a subunit molecular
mass, based on the amino acid sequence [229], of 19824 Da.
All ferritins that have been characterised have the same 24-
subunit structure, although bacterial ferritins have a rather
smaller subunit molecular mass, 15 kDa for the E. coli protein
[230] and 17 kDa for that isolated from Azotobacter species
[231, 2321. Bacterioferritins are also unusual in that they
contain haem, and appear to be identical with cytochrome bl
of E. coli [230, 2331 and with a h-type cytochrome of Azoto-
bacter vinelandii [231, 2341 and of A . chroococcum [232].
The apoferritin shell can aggregate to give dimers, trimers
etc. which are often observed on polyacrylamide gel electro-
phoresis. It is one of the more elementary criteria of the purity
of ferritin preparations that all the protein bands on non-
denaturing gels should stain not only for protein but should
also give a positive Prussian blue reaction. On SDS/polyacryl-
 497

amide gels, purified ferritin preparations frequently present I = horse jpieen A p o l e r r i t i n Trorfi (229)
bands of lower molecular mass than the apoferritin subunit I1 = Hurnari H chairi from (254)
(6 - 8, 11 - 13 and 14- 15 kDa), which appear to result from
proteolytic cleavage of the 19-kDa subunit [235- 2391. Fur- I ~ I = H u r l x i L chain frorii (254)
ther heterogeneity is apparent on SDS gels of mammalian IV = Txlpoio Rerl c e l l A p o f e r r i t i n from (257)
ferritins since in addition to the 19-kDa subunit a second V = Rat Liver L chain f r o m (252)
subunit of 21 kDa is frequently observed; these two subunits
are designated H (heavy, 21 kDa, predominant in heart) and
L (light, 19 kDa, predominant in liver and spleen); tissue
ferritins are assumed to be a mixture of the two
homopolymers, HZ4 and LZ4, and the 23 heteropolymers
HZ3L1-H1LZ3 [240-2461. The multiple bands observed on
isoelectric focussing of tissue ferritins can be explained by this
model if we assume that H and L differ not only in size but
also in their isoelectric points; the ratio of H/L subunits is
both species- and tissue-specific and varies from 1 to 9 in
horse spleen and in human liver [247, 2481 to 8.5 to 1 in horse
heart ferritin [248]. Human H chains are more acidic than L
chains [240], whereas the inverse is the case for horse ferritins
[240, 2481.

Apoferritin: primary structure and comparative studies

The amino acid sequence of horse spleen apoferritin was
first established in 1981 [229] after some 12 years of effort.
Thereafter direct protein sequencing methods established the
primary structure of the human spleen L subunit and of some
70 residues of a minor sequence, assumed to be the H subunit
[249], and of the human liver L chain [250] as well as partial
sequences of human placenta ferritin and of the ferritin of
HeLa cells [251]. cDNA methods have subsequently estab-
lished the sequence of a rat liver L subunit [252], of human
liver H subunit [253,254], human liver L subunit [254], human
lymphocyte H subunit [255] (partial sequence), human mono-
cyte-like cell line U937 L subunit [256] and tadpole red cell
ferritin [257]. The sequences are presented in Fig. 13. The
cDNA studies indicated that both H and L subunits are
encoded by a family of genes in human [253] and rat [258].
However, more recent studies [259,260], including the cloning
and sequencing of the gene coding for human apoferritin H
subunit [260], has established the presence of a number of
pseudogenes. The apoferritin H gene has three introns and its I
exon sequence is identical to that of cDNAs isolated from I!
human liver, lymphocytes, HeLa cells and endothelial cells. I;!
There are at least 15 intronless pseudogenes whose features
1V
suggest that they originated by reverse transcription and inser-
tion; the authors conclude that there is probably only one
gene coding for the human apoferrin H subunit [260]. The
gene for ferritin has been assigned to chromosome 19 [261]
and for the H chain to chromosome 11 [262]. However more
recently ferritin genes have been found on 12 different 151 170 174 178
chromosomes [263]. It is normal, since ferritin is a housekeep-
ing gene, that it would be expressed in the germline of the LGEYLFERL~LKH~
species and so be available for reverse transcription, incorpor- 4----0KH--GDSONES
ation into the genome and transmission to the progeny. The
presence of so many pseudogene copies of the ferritin subunit
tl-----DKH-flGESS
makes the identification of active ferritin genes on chro-
mosomes difficult [259].
Comparison of the amino acid sequences reveals that
horse, human and rat L subunit sequences are considerably
more homologous than are the human L and H sequences:
there are only 14% substitutions between human and horse
L sequences, compared with 45% substitutions for the two
human subunits. It is difficult to assign the tadpole sequence
as H- or L-like since it is almost as similar to the human H Fig. 13. Amino acids sequences of ferritins
498
1
I Table 1. Structural regions of the apoferritin subunit
Fig. 14. Packing of the 24 subunits of the apojerritin molecule. Re-
produced with permission from Ford et al. [264]
Fig. 15. The three-dimensional structure of the subunit of the
apoferritin. Reproduced with permission from Ford et al. [264]
sequence (67% homology) as to the human L chain (59%
homology). The human H subunit has only four amino acid
residues more than its L equivalent and yet has an apparent
molecular mass of 2 kDa more as judged by SDS-PAGE.
The rat L subunit has an eight-residue insertion near the C-
terminus compared to the other two L chains (with which
it has homology of 88% and 82% with horse and human
respectively). Surprisingly direct sequence analysis of peptides
derived from rat liver H subunits revealed that the H-subunit
sequence did not contain this octapeptide. When we compare
all of the available sequences the residues 2 - 8,26 - 39, 106-
114, 122- 131 and 160- 169 show considerable homology
with mostly conservative changes.
Apojerritin: the three-dimensional structure
Horse spleen apoferritin crystallises from aqueous
solutions of CdS04 yielding large octahedral crystals with
unit cell dimension a = 18.48 nm belonging to the cubic space
group F432 [227]. The 24 subunits pack in 4: 3 :2 symmetry,
as illustrated in Fig. 14. The three-dimensional structure has
been solved at a resolution of 0.28 nm, the amino acid se-
quence [229] incorporated into the atomic model and the
structure has been refined to an R value of 0.20 [264, 2651.
The apoferritin subunit (Fig. 15) consists of a bundle of four
long a-helices: A (residues 10-39), B (45-72), C (92- 120)
Reproduced with permission from Rice et al. [265]
Region Residues Region Residues
N-terminal loop 1-9 CD turn 121 - 123
A helix 10-39 D helix 124-155
AB turn 40 - 44 DE turn 156-159
B helix 45 - 72 E helix 160-169
L loop 73-91 T C-terminal tail 170- 174
C helix 92-120
and D (124- 155), with a shorter fifth helix E (160- 169). The
N-terminus, N, iies at the opposite end of the subunit to E,
which is disposed at an acute angle to the main bundle of
helices. The loop L joins helices B and C; together with its
related counterpart in a neighbouring subunit it forms a sec-
tion of antiparallel /3-sheet within the dimer. The pairs of
helices A, B and C, D are connected by short turns such
that these helices are antiparallel. In the view of the subunit
presented in Fig. 15, the helices B and D have one face towards
the inside of the protein shell, and A and C have one face
towards the outside of the molecule. The loop L is also at the
outer surface.
Within the subunit the interior helices and their side chains
interact to form a tightly packed hydrophobic core over a
distance of some 3.5 nm. In the middle of the helical bundle
the character of the interior changes and we find several buried
polar or hydrophilic residues, Tyr-23, Lys-58, Glu-103 and
Glu-I 37, which form a network of hydrogen bonds with a salt
bridge between Lys-58 and Glu-103. The longest helix, D,
extends beyond the main helical bundle and the chain folds
back so that the helix E is disposed at an acute angle to the
helical bundle. It interacts with the subunit through hydro-
phobic interactions involving Phe-166 and Leu-171 and
hydrogen bonds involving Glu-163. The structural regions of
the apoferritin subunit are summarised in Table 1. In the
intact apoferritin molecule there are two types of channels
through which small molecules or ions could gain access to
the central cavity of the molecule. These channels are located
along the threefold and fourfold axes of symmetry and have
very different characters. The six fourfold channels are very
hydrophobic (Fig. 16A). The symmetrical arrangement of
four E helices buries a number of hydrophobic residues,
notably Leu-161, Leu-165 and Leu-169. The channel is 0.3-
0.4 nm in diameter and some 1.2 nm long. In contrast, the
side-chain residues which project into the eight threefold
channels (Fig. 16B) are polar or hydrophilic, notably Asp-
127 and Glu-130. In the crystal these channels are occupied
by metal ions (probably Cd2+ of crystallisation). One is
coordinated to the three Asp residues in the interior of the
channel and the other by the three Glu residues at the outer
surface.
There are two hydrophobic patches on the surface of the
subunit which are buried within the subunit-subunit contacts
of the intact apoferritin shell. The most important involves
residues of helix A (Val-20, Leu-24, Tyr-28, Leu-31) and of
the loop L (Phe-78, Leu-81, Pro-84). This hydrophobic patch,
some 2.2 nm in length, is buried from the solvent by interac-
tion with the equivalent region in a second subunit to form a
twofold symmetry axis. This no doubt explains the observa-
tion that dimers are intermediates in the dissociation of
apoferritin [266].The other hydrophobic region on the surface
of the subunit contains one face of the E helix (Leu-161, Tyr-

Fig. 16. Stereoviews of the apoferritin shell around the threefold ( A ) and,fourfold ( B ) axes. Reproduced with permission from Ford et al. [264]
164, Leu-165, Leu-169 and Leu-154 close to the C-terminus
of the D helix). These residues are buried in the fourfold
channels.
It is clear from X-ray diffraction that the three-dimen-
sional structures of ferritins from horse, human, rat, mouse,
dog and ferret liver and spleen are isomorphous [267].
Separated H and L chains from human, rat and horse ferritins
can form hybrid molecules [268,269] and the physicochemical
properties of horse spleen, liver and heart ferritins, which
contain respectively lo%, 40% and 85% of H subunits are
consistent with the hypothesis that they are hybrids composed
of variable proportions of the two subunit types [248]. Thus,
it would seem that the tertiary and quaternary structure is
conserved to a large extent in both H and L chains. The
difference electron density map (rat liver minus horse spleen)
shows very few features which can be attributed to the eight-
499
residue insertion at residues 158 of the rat protein [270]. This
substitution is in the DE corner, where the electron density is
weak, perhaps on account of its flexibility. There is however
a region of negative density near residue 48, which is consistent
with the substitution of Cys in the horse protein by Gly in rat
liver and with the loss of a bound Cd2+ ion.
Mouse ferritin exhibits structural homology with horse
spleen ferritin as judged by X-ray diffraction patterns [270]
although it is reported to contain almost exclusively heavy
chains [271]. It is suggested [270] that insertions in the se-
quence may occur without disrupting the quaternary structure
of the apoferritin: known sites of insertion are at the N-
terminus and the C-terminus of the human H chain and in
the DE turn in the rat liver L chain (see Fig. 13).
When the different apoferritin sequences presented in
Fig. 13 are compared with the horse spleen subunit structure,
500
it is clear that residues which are buried within the subunit
are much more conserved than are residues exposed on the
outer or inner surfaces. Thus, over half of the 60 buried
residues of the subunit are invariant and of the remainder
two-thirds of the replacements are conservative. About a third
of the external residues are invariant, and less than a third
of the residues on the internal surface are conserved. In all
apoferritin subunits the sequence of residues 122- 131 is
conserved (with the exception of the substitution of Leu-129
by Ile); this sequence includes Asp-127 and Glu-130, potential
metal-binding sites at the exterior and interior of the threefold
channels.
In the horse spleen apoferritin molecule there are a great
number of hydrogen bonds and salt bridges both within the
subunit and at the interfaces between subunits, which most
probably contribute to the considerable thermostability of the
molecule. Almost half of these interactions are conserved in
all of the sequences and most of the others are replaced by
alternative interactions. However, in the centre of the subunit,
in a region rich in polar residues and flanked on either side by
two densely packed hydrophobic regions, there is considerable
variability, such as the change of Lys-58 (which forms a salt
bridge with Glu-103 that certainly contributes in a important
way to the stability of the subunit) to Glu in the human H
sequence. Conservation of residues in potential metal binding
sites is discussed below. Although ferritin from E. coli has
been crystallised in the same space group as horse spleen
ferritin [230], a detailed structure is not yet available. However
partial amino acid sequence data on this protein suggests that
it has no structural homology with mammalian ferritins [272].
Iron deposition in ferritin
We can imagine at least two totally different mechanisms
for iron incorporation into ferritin, namely the assembly of
apoferritin subunits around preformed micellar iron cores or
else the penetration of Fe(I1) into the apoferritin shell and its
subsequent oxidation, hydrolysis and polymerisation to give
the ferric oxyhydroxide polymer.
The former was suggested by Saltman and his colla-
borators [273] on the basis of studies on the hydrolysis and
polymerisation of Fe(II1) chelates. They synthesized a poly-
mer of approximate composition Fe403(OH)4 . (NO,),
. 1.5 H 2 0 which not only had the same diameter, 7.0 nm, as
the ferritin iron core, but which also had similar spectroscopic
properties [213, 2741. They showed that when the cor-
responding ferric citrate micelle was placed in a solution of
noncrystalline apoferritin the synthetic core was surrounded
by the protein subunits to give a product similar to ferritin in
its gross morphology [273]. However, since the biosynthesis
of apoferritin precedes that of ferritin it is unlikely that such
a mechanism is involved.
Ferritin-like products are formed when apoferrin is in-
cubated with ferrous salts in the presence of appropriate
oxidants [276-2811. Neiderer [279] advanced the hypothesis
that Fe(I1) ions could penetrate the protein shell of the
apoferritin molecule where their oxidation would be catalysed
by the protein: the nascent ferric ions would form an in-
tramolecular precipitate of FeO . OH which would soon be
too big to escape. This view has received considerable support
over the years, and a certain number of facts have been estab-
lished, even if we have not yet identified in the three-dimen-
sional structure of the protein the precise sites involved.
Kinetic studies using ferrous salts (usually ferrous ammonium
sulphate) with an appropriate oxidant (usually molecular oxy-
gen) show that iron incorporation is a complex process,
characterised by two distinct phases [280,282- 2841. The first
slow stage corresponds to initial iron binding and oxidation,
implying specific sites on the apoferritin molecule. This is
often referred to as the nucleation phase. The subsequent
growth of the iron micelle is accompanied by further oxida-
tion, hydrolysis and polymerisation. This second phase may
occur on the surface of the micelle, and hence no longer
require catalysis by the protein [280], or may still necessitate
an active role for the protein [281]. The initial phase is at
least second order in iron [283-2851, and this together with
inhibition studies [284- 2861 suggests that the first steps in
iron binding and oxidation involve a bimetallic site [287,288].
Carboxylate functions are clearly implied in the catalysis of
iron oxidation as indicated by chemical modification studies
[289] and by EXAFS [290]. An EPR study of iron accumula-
tion in apoferritin has identified what appears to be a spin-
coupled Fe(I1)-Fe(II1) dimer with a net electron spin of 1/2
[291]. The binding stoichiometries of Mn(II), VO(1V) and
Cd(I1) to apoferritin are close to 0.3 and 0.7 metal ion per
subunit [292], close to the values of 0.33 and 0.67 predicted
for the binding of one and two metal ions respectively per
three subunits. This would be consistent with the binding of
one or two metal ions in each of the eight hydrophilic channels
along the threefold axes: the X-ray crystallographic study
shows [293] that cadmium binding sites are found on the
outside and inside of these channels (constituted respectively
by three Glu-130 residues and three Asp-127 residues from
symmetry-related subunits).
We should however note here that if Fe(I1) appears to be
more readily assimilated by apoferritin in vivo than Fe(II1)
[294, 2951, the iron which is released from transferrin within
the cell, as we have seen above, is ferric and must thus be
reduced prior to its incorporation into the storage protein.
An alternative might be that in vivo iron is present as a low-
molecular-mass polynuclear form, as has been proposed for
the iron utilised for haem synthesis [296,297] and that it is in
this form that it is incorporated into apoferritin.
Storage iron mobilisation
In vitro iron can be mobilised from ferritin by direct chela-
tion of Fe(II1) or by reduction in the presence of an ap-
propriate chelator of Fe(I1). Iron release is rather slow both
for a number of biological chelators [298-3011 and with
synthetic chelators of Fe(II1) [300, 3021 or with microbial
siderophores [303- 3051. However much more rapid rates
were observed for the chelation of ferritin iron under argon by
dihydrolipoate and dihydrolipoamide [306]. In general much
more rapid rates of iron release are observed with reducing
agents such as dithionite, dihydroflavins or thioglycollate
[307- 3111in the presence of a$-bipyridyl. Mobilisation rates
are consistently more rapid at acidic than at neutral pH values
[309], and it has been proposed that specific interactions occur
between products or educt and the interfacial iron(II1)
hydroxide of the ferritin core [311]. Although it has been
suggested that dihydroflavins must penetrate into the interior
of the protein shell in order to reduce iron [309], the recent
study found no evidence of hindered shell penetration [311].
This is consistent with the observation that a number of quite
large molecules and ions can gain access to the interior of the
ferritin molecule [204, 219, 312, 3131. Reductive mobilisation
Educt: a substance separated unchanged from another sub-
stance; distinguished from product.

of ferritin iron by superoxide [314-3171 and by ascorbate in
the presence of oxygen [318] have also been reported: the
former is extremely slow, while the latter is attributed to the
monodehydroascorbate radical.
We might now pose the question as to what is the chemical
nature of the ferritin iron mobilising agent in vivo. Of the
biological reductants, dihydroflavins would seem to be
unlikely on account of the extremely low intracellular concen-
tration of free flavins and the absence of evidence in animal
cells of a flavin reductase. Likewise the concentrations of
lipoate and its derivatives within cells are many orders of
magnitude below those used in the iron mobilisation experi-
ments. Superoxide, whether produced by xanthine oxidase in
its NAD-dependent dehydrogenase form or by haemoglobin
or myoglobin in the presence of oxygen seems unlikely not
only on account of the high levels of superoxide dismutase
within cells, but also on account of the potential danger to
the cell of Fenton chemistry, as discussed previously.
Not only is the nature of the iron mobilising agent un-
known, we do not know with any certainty where storage is
mobilised within the cell. However, since we find that ferritin
and haemosiderin iron is more readily mobilised at acidic pH
values than at neutrality both by reductants and by chelators
[311] (and R. R. Crichton and collaborators, unpublished
observations), lysosomal haemosiderin iron might be a good
target for therapeutic chelators in the treatment of secondary
iron overload.
Intracellular iron metabolism
Within the cell, iron is present in several forms and is
distributed within different intracellular compartments. We
have already mentioned the storage forms ferritin (in large
part in the cytoplasm) and haemosiderin (in secondary
lysosomes). There are haem proteins, iron-sulphur proteins
and proteins such as ribonucleotide reductase and the aromat-
ic amino acid hydroxylases which contain non-haem iron
which is not in an iron-sulphur cluster [319]. There is however
an intermediate pool of chelatable or transit iron the
appearances of which are ‘somewhat like the Loch Ness
monster, only to disappear from view before its presence, or
indeed its nature, can be confirmed’ [320]. While it has been
claimed that this might represent up to 20% of intrahepatic
iron [321], this seems rather unlikely and it is more probable
that this pool represents only a very small amount of iron. It
is presumably composed of iron (ferrous of ferric) complexed
by low-molecular-mass chelators and may correspond to the
low-molecular-mass polynuclear forms of ferric chelates
found in the course of controlled hydrolysis and polymerisa-
tion of these latter [4, 2961. This cytosol iron pool is often
referred to as the transit iron pool based on the assumption
that it represents iron in transit between transferrin and
ferritin [322].
Intracellular iron metabolism. not only involves iron
uptake by the cell from transferrin, but also the supply of iron
within the cell for synthesis of iron-containing proteins and
the mechanisms which allow iron to be excreted from the cell.
We have already considered the first point and we address the
others in the paragraphs which follow.
The incorporation of iron into non-haem iron proteins is
very poorly studied and we will not consider it here. How-
ever the terminal step in haem biosynthesis, catalysed by
ferrochelatase, is somewhat better established. This enzyme,
located in the matrix face of the inner mitochondrial mem-
brane [323], incorporates Fe(I1) into protoporphyrin IX to
501
form haem. The source of iron for ferrochelatase might be
transferrin, ferritin or the transit iron pool. While it has been
shown in vitro that transferrin can readily supply iron for
haem synthesis by isolated mitochondria [324- 3261, in view
of the transferrin-to-cell cycle presented above, it is difficult
to see how a transferrin molecule bearing iron could come
into physical contact with a mitochondrion. It has been
suggested that an FMN-dependent ferriductase associated
with the mitochondria could enable electrons to be siphoned
from the respiratory chain to reductively mobilise ferritin iron
for haem synthesis [327- 3301.
This does not seem to be likely [297] since the proposed
specificity of interaction between ferritin and the mito-
chondrion is not observed when homologous and
hererologous systems are compared, no specific binding of
ferritin to the mitochondrion is observed and the rate of haem
synthesis is essentially identical for mitoplasts and
mitochondria. This leaves the possibility that the presumed
low-molecular-mass polynuclears constituting the transit iron
pool [4,296] are the principal source of iron for ferrochelatase
together with cytosol ferritin. There are also reports in the
literature of a pool of non-haem, non-iron-sulphur mito-
chondrial iron which represents about 25% of total
mitochondrial iron and which could function as a proximate
iron donor to ferrochelatase in vivo [331- 3331.
At present we know very little about the pathways of
storage iron mobilisation from cells. In normal human sub-
jects some 40 mg of tissue iron is mobilised per day, 80% due
to the recycling of iron derived from the catabolism of effete
red blood cells by the macrophages of the reticuloendothelial
system to the erythroid bone marrow for haemoglobin syn-
thesis [143]. Although the presence of specific receptors for
apotransferrin has been reported on activated peritoneal
macrophages [334], the presence of apotransferrin in the
culture medium does not appear to be essential for the excre-
tion of iron by the cells [335]. It seems that iron is released
from macrophages as newly synthesized macrophage ferritin
which, in the culture medium, releases its iron to
apotransferrin [335]. This transfer from ferritin to apo-
transferrin is stimulated by low-molecular-mass serum factors
(R. R. Crichton and Y. Jin, unpublished results).
In conclusion, we may compare intracellular iron metab-
olism with a symphony orchestra: we can, in the absence of
the conductor, identify the different instruments but not the
role that they will play. From the view point of the brass
section, it seems clear that the horns and trumpets of cytosol
ferritin have the lion’s share of the symphony to be performed,
with a supporting role for the lysosomal trombones and tubas
of haemosiderin. In the string section it seems obvious that
the violins of mitochondrial ferrochelatase reinforced by the
counterpoint of the altos, cellos and double bases of haem
degradation have the principal role to play. The sharply in-
flected tones of the flutes and clarinets of the transit iron pool
are supported by the bassoons and oboes of the Schnei-
der formation who have surreptitiously introduced low-
molecular-mass polynuclears. And in the face of all this
confusion, the transferrin percussion section resonates all of
its forces against the cell membrane, uncertain whether they
want to get out or to get in . . .
Once the conductor has taken his place in the middle of
this chaos, we begin to hear clearly, and we realise that the
principal role will be given to each section of the orchestra as
a function of the music to be played. And that is probably
how intracellular iron metabolism is regulated: not by a pre-
ponderant role of any one of the protagonists but by the
harmonious repartition of their contributions in order to
assure the harmony, homogeneity and homeostasis of the
ensemble.
Epigram
While we have learned much in the last few years about
iron transport and storage, there remains an important
number of fundamental questions as yet unanswered. What
mechanism regulates the iron requirements of a given cell,
whether by the secretion of siderophores or by the number of
transferrin receptors expressed at the cell surface? How
is storage iron assimilated and released? What are the
mechanisms involved in intracellular iron metabolism, and
how is iron released from cells; the transferrin-cell cycle is
well established, but how does iron get out of the cellular
prison in which it is incarcerated? Since we wish to end with
an epigram, we can conclude that, the more we know about
iron storage and transport - the more we need to know!
REFERENCES
1. Aisen, P. (1977) Ciba Found. Symp. 51, 1 - 14.
2. Cotton. F. A. & Wilkinson. G. (1980) in Advanced inorganic
~I
chemistry 4th edn, pp. 750-751, Wiley-Interscience, “New
York.
3. Hill, H. A. 0.(1982) in The biologicalchemistry of iron (Dunford,
H. B., Dolphin, D., Raymond, K. N . & Seiker, L., eds) pp. I -
12, Reidel, Dordrecht.
4. Schneider, W. & Schwyn, B. (1987) in Aquatic surface chemistry
(Stumm, W., ed.) Wiley-Intersciences, New York, in the press.
5. Neilands, J. B. (1981) Annu. Rev. Biochem. 50, 715-731.
6. Haber, F. & Weiss, J. (1934) Proc. R. Soc. Lond. A 147, 332-
337.
I. Wrigglesworth, J. M. & Baum, H . (1980) in Iron in biochemistry
and medicine, 2nd edn (Jacobs, A. & Worwood, M., eds)
pp. 29-86, Academic Press, London.
8. Neilands, J. B. (1981) Annu. Rev. Nutr. 1, 27-46.
9. Crosa, J. H. & Hodges, L. L. (1981) Infect. Immunol. 31, 223-
227.
10. Walter, M. A., Potter, S. A. & Crosa, J. H. (1983) J . Bacteriol.
156,880-887.
11. Neilands, J. B. (1952) 1. Am. Chem. Soc. 74,4846-4847.
12. Keller-Scherlein, W., Prelog, V. & Zahner, H. (1964) Fortschr.
Chem. Org. Naturst. 22, 279-333.
13. Lankford, C. E. (1973) Crit. Rev. Microbiol. 2, 273-331.
14. Snow, G. A. (1970) Bacteriol. Rev. 34, 99- 125.
15. Wayne, R. & Neilands, J. B. (1975) J . Bacteriol. 121, 497-503.
16. Jacobs, A., White, G. P. & Tait, G. H. (1977) Biochern. Biophys.
Res. Commun. 74, 1626- 1630.
17. Raymond, K. N. & Carrano, C. J. (1979) Acc. Chem. Res. 12,
183- 196.
18. Neilands, J. B., Peterson, T. & Leong, S. A. (1980) in Inorganic
chemistry in biology andmedicine (Martell, A. E., ed.) pp. 263 -
278, American Chemical Society, Washington DC.
19. Raymond, K. N., Muller, G. & Matzanke, B. F. (1984) Top.
Curr. Chem. 123, 50-102.
20. Winkelman, G. (1979) FEBS Lett. 97, 43-46.
21. Winkelman, G. & Braun, V. (1981) FEMS Microbiol. Lett. 11:
237-241.
22. Muller, G., Isowa, Y. & Raymond, K . N. (1985) J . Biol. Chem.
260, 13921 - 13926.
23. Neilands, J. B., Erickson, T. J. & Rastetter, W. H. (1981) J . Biol.
Chem. 256,3831 - 3832.
Definition: ‘Any terse, witty, pointed statement often anti-
thetical’. Example: ‘Experience is the name everyone gives to his
mistakes’.
24. Frost, G. E. & Rosenberg, H. (1973) Biochim. Biophys. Acta
330,90-101.
25. Neilands, J. B. (1982) Annu. Rev. Microbiol. 36, 285-309.
26. Brdun, V. (1985) in The enzymes of biological membranes vol. 3,
(Martonosi, A. N., ed.) pp. 617-652, Plenum Press, New
York.
27. Nikaido, H. (1979) in Bacterial outer membranes (Inouye, M.,
ed.) pp. 361 -407, Wiley, New York.
28. Luckey, M., Pollack, J . R., Wayne, R., Ames, B. N. & Neilands,
J. B. (1972) J. Bacteriol. 111, 731 -738.
29. Wayne, R. & Neilands, J. B. (1975) J. Bacteriol. 121, 497-503.
30. Guterman, S. K. (1973) J . Bacteriol. 114, 1217-1224.
31. Krone, W. J. A., Stegehuis, F., Konigstein, G., de Graaf, F.
K. & Oudega, B. (1985) FEMS Microbiol. Lett. 26, 153-
161.
32. Matzanke, B. F., Muller, G. I. & Raymond, K. N. (1984) Bio-
chem. Biophys. Res. Comrnun. 121,922-930.
33. Fecker, L. & Braun, V. (1984) J . Bacteriol. 156, 1301-1314.
34. Hantke, K. (1985) in Proteins of iron storage and transport (Spik,
G., Montreuil, J., Crichton, R. R. & Mazurier, J., eds)
pp. 231 -243, Elsevier, Amsterdam.
35. Ratledge, C., Patel, P. V. & Mundy, J. (1982) J . Gen. Microbiol.
128, 1559-1565.
36. Emery, T. (1971) Biochemistry 10, 1483- 1488.
31. Carrano, C. J. &Raymond, K . N. (1978) J . Bacteriol. 136,69-
74.
38. O’Brien, I. G., Cox, G. B. &Gibson, F. (1971) Biochim. Biophys.
Acta 237, 531-549.
39 Langman, L., Young, 1. G., Frost, G. E., Rosenberg, H. &
Gibson, F. (1972) J . Bacteriol. 112, 1142-1149.
40 Porra, R. J., Langman, L., Young, I. G. & Gibson, F. (1972)
Arch. Biochem. Biophys. 153, 74-78.
41 Emery, T. (1976) Biochemistry 15,2723-2728.
42 Lodge, J. S., Gaines, C. G., Arceneaux, J. E. L. & Byers, B. R.
(1980) Biochem. Biophys. Res. Commun. 97,1291 - 1295.
43 Coderre, P. E. & Earhart, C. F. (1984) FEMS Microbiol. Lett.
25, 111 -116.
44 Fleming, T. P., Nahlik, M. S., Neilands, J. B. & McIntosh, M.
A. (1985) Gene 34,47-54.
45. Straka, J. G. & Emery, T. (1979) Biochim. Biophys. Acta 569,
277 - 286.
46. Brown, K. A. & Ratledge, C. (1975) FEBS Lett. 53,262-266.
47. Ernst, J. F. & Winkelmann, G. (1977) Biochim. Biophys. Acta
500, 27 -41.
48. Hartmann, A. & Braun, V. (1980) J. Bacteriol. 143, 246-255.
49. Braun, V., Brazel-Faisst, C. & Schneider, R. (1984) FEMS
Microbiol. Lett. 21, 99- 103.
50. Osborne, T. B. & Cambell, G. F. (1900) J . Am. Chem. Soc. 22,
422 -426.
51. Alderton, G., Ward, W. H. & Ferold (1946) Arch. Biochem.
Biophys. 11, 9-13.
52. Schade, A. L. & Caroline, L. (1944) Science (Wash. DC) 100,
14-15.
53. Schade, A. L. & Caroline, L. (1946) Science (Wash. DC) 104,
340 - 341.
54. Laurell, C. B. &Ingelman, B. (1947) Acta Chem. Scand. 1,770-
776.
55. Holmberg, C. G. & Laurell, C. B. (1947) Acfa Chem. Scand. 1,
944-950.
56. Schade, A. L., Reinhart, R. W. & Levy, H. (1949) Arch. Biochem.
Biophys. 20, 170- 172.
57. Surgenor, D. M., Koechlin, B. A. & Strong, L. E. (1949) J. Clin.
Invest. 28, 73 -96.
58. Montreuil, J., Tornelat, J. & Mullet, S. (1960) Biochim. Biophys.
Acta 45,413-421.
59. Masson, P. L. & Heremans, J. F. (1968) Eur. J . Biochem. 6,
579 - 584.
60. Aisen, P. & Listowsky, I. (1980) Annu. Rev. Biochem. 49, 357-
393.
61. Chasteen, N. D. (1977) Coord. Chem. Rev. 22,l-36.
62. de Lucas, L. J., Suddath, F. L., Gams, R. A. & Bugg, C . E.
(1978) J. Mol. Bid. 123, 285-2286,

63. Al-Hilal, D., Baker, E., Carlisle, C. H., Gorinsky, B., Horsburgh,
R. C., Linley, P. F., Moss, D. S., Schneider, H. & Stimpson,
R. (1976) J . Mol. Bid. 108, 255-257.
64. Gorinsky, B., Horsburgh, C., Lindley, P. F., Moss, D., Parkcr,
M. &Watson, J. L. (1979) Nature (Lond.) 281, 157-158..
65. Baker, E. N. & Rumball, S. V. (1977) J . Mol. Biol. 111, 207-
210.
66. Rossencu-Motreff, M. Y., Soctewey, F., Lamote, R. & Peeters,
H. (1971) Biopolymers 10, 1039-1048.
67. Mac Gillevray, R. T. A,, Mendez, E., Sinha, S. K., Sutton, M.
R., Linebach-Zins, J. & Brew, K. (1982) Proc. Nut1 Acad. Sci.
U S A 79, 2504-2508.
68. Mac Gillivray, R. T. A., Mendez, E., Shewale, J. G., Sinha, C.
K., Linebach-Zins, J. & Brcw, K. (1983) J . Biol. Chem. 258,
3543 - 3553.
69. Uxan, G., Frain, M., Park, I., Besmond, C., Haessen, G., Trepat,
J. S., Zakin, M. M. & Kahn, A. (1984) Biochem. Biophys. Res.
Commun. l l Y , 273-281.
70. Yang, F., Lum, J. B., McGill, J. R., Moore, C. M., Naylor, S.
L., Van Bragt, P. H., Baldwin, W. D. & Bowman, B. H. (1984)
Proc. Natl Acad. Sci. USA 81,2752-2756.
71. Jeltsch, J. M. & Chambon, P. (1982) Ezcr. J . Biochem. 122,29 1 -
296.
72. Williams, J., Elleman, T. C., Kingston, J. B., Wilkins, A. G. &
Kuhn, K. A. (1982) Eur. J. Biochem. 122,297-303.
73. Metz-Boutigue, M.-H., Jolles, J., Mazurier, J., Schoentgen, F.,
Legrand, D. S., Spik, G., Montreuil, J. & JollZs, P. (1984) Eur.
J . Biochem. 145, 659-676.
74. Staden, R. A. (1982) Nucleic Acids Res. 10, 2951 -2961.
75. Mac Gillivray, R. T. A,, Mendez, E. & Brew, K. (1977) in
Proteins ufiron metabolism (Brown, E. B., Aisen, P., Fielding,
J. & Crichton, R. R., eds) pp. 131-142, Grune & Stratton,
New York.
76. Mazurier, J., Metz-Boutigue, M. H., Jollks, J., Spik, G.,
Montreuil, J. & Jollks, P. (1983) Experientia (Basel) 39, 135-
141.
77. Smith, T. F. & Waterman, M. S. (1981) Adv. Appl. Math. 2,
482 - 489.
78. Spik, G., Bayard, B., Fournet, B., Strccker, G., Bouquelet, S. &
Montreuil, J. (1975) FEBS Lett. 50, 296-2299,
79. Dorland, L., Haverkamp, J., Schut, B. L., Vliegenthart, J. F.
G., Spik, G., Strecker, G., Fournet, B. & Montreuil, J. (1977)
FEBS Lett. 77, 15 - 20.
80. Karlsson, K. A., Pascher, I., Samuelsson, B. E., Finne, J.,
Krusius, T. & Rauvala, H. (1978) FEBS Lett. 94,413-417.
81. Spik, G., Debruyne, V. & Montreuil, J. (1982) Falk Symp. 34,
477-483.
82. Marz, L., Hatton, M. W. C., Berry, L. R. & Regoeczi, E. (1982)
Can. .I. Biochem. 60, 624- 630.
83. Spik, G . , Strecker, G., Fournet, B., Bouquelet, S., Montreuil,
J., Dorland, L., Van Halbeek, H. &Vliegenthart, J. F. G. (1982)
Eur. J . Biochem. 121,413-419.
84. Dorland, L., Haverkamp, J., Vliegenthart, J. F. G., Spik, G.,
Fournet, B. & Montreuil, J. (1979) Eur. J . Biochem. 100,569-
574.
85. Williams, J. (1982) Trends Biochem. Sci. 7, 394-397.
86. Williams, J., Grace, S. A. & Williams, J. M. (1982) Biochem. J .
201, 417-419.
87. Warner, R. C . & Weber, I. (1953) J . Am. Chem. Soc. 75, 5094-
5101.
88. Aasa, R., Malmstrom, B. G., Saltman, P. & Vanngird, T. (1963)
Biochim. Biophys. Acta 75, 203-222.
89. Aisen, P., Leibman, A. & Reich, H. A. (1966) J . Biol. Chem.
241, 1666-1671.
90. Wenn, R. V. & Williams, J. (1968) Biochem. J . 198, 69-74.
91. Princiotto, J . V. & Zapolski, E. J. (1968) Nature (Lond.) 255,
87-88.
92. Lestas, A. N. (1976) Br. J . Huematol. 32, 341 -350.
93. Butterworth, R. M., Gibson, J . F. & Williams, J. (1975) Biochem.
J . 149, 559-563.
94. Makey, D. G. & Seal, U. S. (1976) Biochim. Biophys. Acta 453,
250 - 256.
503
95. Aisen, P., Leibman, A. & Zweier, J. (1978) J . Biol. Chem. 253,
1930-1937.
96. Aisen, P. & Leibman, A. (1978) in Trunsport byproteins(Blauer,
G. & Sund, H., eds) pp. 277- 294, W. de Gruyter & Co., Berlin,
New York.
97. Aasa, R. (1972) Biochem. Biophys. Rex Commun. 49, 806-
812.
98. Rogers, T. B., Gold, B. A. & Feeney, R. E. (1977) Biochemistry
16,2299-2305.
99. Alsaadi, B. M., Williams, R. J. P. & Woodworth, R. C. (1981)
J . Inorg. Biochem. 15, 1-6.
100. Pecorara, V. L., Harris, W. R., Carrano, C. J . & Raymond, K.
N. (1981) Biochemistry 20, 7033-7039.
101. Williams, J. (1982) Biochem. J . 201, 647-651.
102. Koenig, S. H. & Schillinger, W. E. (1969) J . Bid. Chem. 244,
6520-6526.
103. Harris, D. C. & Gelb, M. H. (1980) Biochim. Biophys. Acta 623,
1-9.
104. Rogers, T. B., Buerresen, T. & Feeney, J. (1978) Biochemistry
17,1105-1109.
105. Chasteen, N. D. (1983) Trends Biochem. Sci. 8, 272-275.
106. Harris, D. C. & Aisen, P. (1973) Biochim. Biophys. Actu 329,
156- 158.
107. Gaber, B. P. & Aisen, P. (1970) Biochim. Biophys. Aciu 221,
228 - 233.
108. Bates, G. W. & Schlabach, M. R. (1973) J . Biol. Chem. 248,
3228 - 3232.
109. Workman, E. F. Jr, Graham, G. & Bates, G. W. (1975) Biochim.
Biophys. Acta 399, 254 - 264.
310. Batcs, G. W. & Wernicke, J. (1917) J . Biol. Chem. 246, 3679-
3685.
111. Bates, G. W., Billups, C. & Saltman, P. (1967) J . Biol. Chem.
242, 2810-2815.
112. Aisen, P., Aasa, R., Malmstrom, B. G. & Vanngard, T. (1967)
J . Bid. Chem. 242, 2484 - 2490.
113. Woodworth, R. C., Virkaitis, L. M., Woodbury, R. G. & Fava,
R. A. (1975) in Proteins of iron storage and transport in hiochem-
istry and medicine (Crichton, R. R., ed.) pp. 39 - 50, North-
Holland, Amsterdam.
114. Bates, G . W. & Schlabach, M. R. (1975) in Proteins of iron
storage and transport in biochemistry m d medicine (Crichton,
R. R., cd.) pp. 51 -58, North-Holland, Amsterdam.
1 15. Masson, P. L. & Heremans, J. (1968) Eur. J . Biochem. 6 , 579 -
584.
116. Carrano, C. J. & Raymond, K . N. (1979) J . Am. Chem. Soc.
101, 5401 - 5404.
117. Pecoraro, V. L., Weitl, F. L. & Raymond, K. N. (1981) .I. Am.
Chem. Soc. 103, 5133-5140.
11 8. Hallberg, L. & Hedenberg, L. (1 965) Scand. J . Haemutol. 2,67 -
75.
119. Morgan, E. H. (1971) Biochim. Biophys. Acfu244, 103-116.
120. Pollack, S., Vanderhoff, G. & Lasky, F. (1971) Biochinz. Biophys.
Act0 497, 481 -487.
121. Morgan, E. H. (1977) Biochim. Biophys. Acta 449, 169-177.
122. Rodgers, S. J. & Raymond, K . N. (1983) J . Med. Chem. 26,
439 -443.
123. Cowart, R. E., Kojima, N. & Bates, G. W. (1982) J . Biol. (Miem.
257,7560-7565.
124. Kojima, N. & Bates, G. W. (1979) J . Bid. Chem. 254, 8847-
8854.
125. Sommer, J. H., O'Hara, P. B., Jonah, C. D. & Bersohn, R. (1982)
Biochim. Biophys. Acta 703, 62 -68.
126. Jandl, J. M. & Katz, J. H. (1963) J . Clin. Invest. 42, 314-326.
127. Iacopetta, B. J., Morgan, E. H. & Yeoh, G. C. T. (1982) Biochim.
Biophys. Acta 687, 204-210.
128. Mattia, E., Rao, K., Shapiro, D. S., Sussman, H. & Klausner,
R. D. (1984) J . Biol. Chem. 259, 2689-2692.
129. Trowbridge, I. S. & Omary, M. B. (1981) Proc. Natl Acud. Sci.
USA 78, 3039 - 3043.
130. Sutherland, D. R., Delia, D., Schneider, C., Newman, R. A.,
Fcmshcad, J. & Greaves, M. F. (1981) Proc. Nail Acad. Sci.
USA 78, 4535-4519.
131. Haynes, B. F., Hemler, M., Cotner, T., Mann, D. L., Eisenberth,
G. S., Strominger, J. L. & Fauci, A. S. (1981) J . Immunol. 127,
347 - 351.
132. Newman, R., Schneider, C., Sutherland, R., Vodinelich, L. &
Greaves, M. F. (1982) Trends Biochem. Sci. 7, 397-400.
133. Seligman, P. A,, Schleicher, R. B. & Allen, R. H. (1979) J . Biol.
Chem. 254,9943 - 9946.
134. Hu, H. Y. Y. & Aisen, P. (1978) J . Supramol. Struct. 8,349-360.
135. Fernandez-Pol, J. A. & Klos, D. J. (1980) Biochemistry 19,
3904-3912.
136. Schneider, C., Sutherland, R., Newman, R. A. & Greaves, M.
F. (1982) J . Biol. Chem. 257,8516-8522.
137. Omary, M. B. & Trowbridge, I. S. (1981) J . Biol. Chem. 256,
471 5 -471 8.
138. Goodfellow, P. N., Banting, G., Sutherland, D. R., Greaves, M.
F., Solomon, E. &Povey, S. (1982) Somat. CellGenet. 8,197-
206.
139. Enns, C. A,, Suomalainen, H. A., Gebhardt, J. E., Schroder,
J. & Sussman, H. H. (1982) Proc. Natl Acad. Sci. USA 79,
3241 - 3245.
140. Schneider, C., Kurkinen, M. & Greaves, M. F. (1983) EMBO J .
2,2259 - 2265.
141. Plowman, G. D., Brown, J. P.,'Enns, C. A,, Schroder, J.,
Nikinma, B., Sussman, H. H., Hellstrom, K. E. & Hellstrom,
I. (1983) Nature (Lond.) 303, 70-72.
142. Goubin, G., Goldman, D. S., Luce, J., Neiman, P. E. & Cooper,
G. M. (1983) Nature (Lond.) 302, 114-117.
143. Awai, M. & Brown, E. B. (1963) J. Lab. Clin. Med. 61, 363-
368.
144. Bothwell, T. H., Charlton, R. W., Cook, J. D. & Finch, C. A.
(1979) in Iron metabolism in man, p. 334, Blackwell, Oxford.
145. Baker, E. (1977) in Ciba Found. Symp. 51, 365-373.
146. Morgan, E. H. & Baker, E. (1969) Biochim. Biophys. Acta 184,
442 -454.
147. Baker, E. &Morgan, E. H. (1968) Biochemistry 8, 1133-1141.
148. Morgan, E. H. & Appleton, T. C. (1969) Nature (Lond.) 223,
1371- 1372.
149. Sullivan, A. L., Grasso, J. A. & Weintraub, L. R. (1976) Blood
47,133-143.
150. Hemmaplardh, D. & Morgan, E. H. (1977) Br. J. Haematol. 36,
85 - 96.
151. Schneider, Y.-J., Tulkens, P., de Duve, C. & Trouet, A. (1979)
J . Cell Biol. 82, 466 -474.
152. Octave, J. N., Schneider, Y. J., Hoffmann, P., Trouet, A. &
Crichton, R. R. (1979) FEBS Lett. 108, 127-130..
153. Octave, J. N., Schneider, Y. J., Crichton, R. R. & Trouet, A.
(1983) Eur. 1.Biochem. 115,611 -618.
154. Octave, J. N., Schneider, Y. J., Hoffmann, P., Trouet, A. &
Crichton, R. R. (1982) Eur. J. Biochem. 123,235-240.
155. Karin, M. & Mintz, B. (1981) J. Biol. Chem. 256, 3245-3252.
156. Paterson, S. & Morgan, E. H. (1980) J . Cell Physiol. 105,489-
502.
157. Morgan, E. H. (1981) Biochim. Biophys. Acta 642, 119-134.
158. Light, A. & Morgan, E. H. (1982) Scand. J . Haemutol. 28,205-
214.
159. Armstrong, N. J. & Morgan, E. H. (1983) Biochim. Biophys.
Acta 762, 175- 186.
160. Iacopetta, B. J. & Morgan, E. H. (1983) J. Biol. Chem. 258,
9108-9115.
161. Iacopetta, B. J. & Morgan, E. H. (1983) Eur. J . Cell Biol. 32,
17-23.
162. Klausner, R. D., Van Renswoude, J., Ashwell, G., Kempf, C.,
Schechter, A. N., Dean, A. & Bridges, K. R. (1983) J. Biol.
Chem. 258,4715 -4724.
163. Ciechanover, A,, Schwartz, A. L., Dautry-Varsat, A. & Lodish,
H. F. (1983) J . Biol. Chem. 258,9681 -9689.
164. Van Renswoude, J., Bridges, K. R., Harford, J. B. & Klausner,
R. D. (1982) Proc. Natl Acad. Sci. USA 79, 6186-6190.
165. Enns, C. A,, Larrick, J. W., Suomalainen, H., Schroder, J. &
Sussman, H. H. (1983) J . Cell Biol. 97, 579-585.
166. Hopkins, C. R. & Trowbridge, I. S. (1983) J . CellBiol. 97, 508-
521.
167. Lamb, J. E., Ray, F., Ward, J. H., Kushner, J. P. & Kaplan, J.
(1983) J. Biol. Chem. 258,8751 -8758.
168. Rao, K., van Renswoude, J., Kempf, C. & Klausner, R. D.
(1983) FEBS Lett. 160, 213-216.
169. Hopkins, C. R. (1983) Cell35, 321-330.
170. Morgan, E. H. (1983) Biochim. Biophys. Acta 762,498-502.
171. Dautry-Varsat, A., Ciechanover, A. & Lodish, H. F. (1983) Proc.
Natl Acad. Sci. U SA 80,2258 -2262.
172. Klausner, R. D., Ashwell, G., van Renswoude, J., Harford, J.
B. & Bridges, K. R. (1983) Proc. Nut1 Acad. Sci. USA 80,
2263 - 2266.
173. Booth, A. G. &Wilson, M. J. (1981) Biochem. J . 196,355-362.
174. Bleil, J. D. & Bretscher, M. S. (1982) EMBO J . 1, 351 -355.
175. Pearse, B. M. F. (1982) Proc. Nut1 Acad. Sci. USA 79, 451-
455.
176. Morgan, E. H., Smith, G. D. & Peters, T. J. (1986) Biochem. J.
237, 163-173.
177. Sibille, J. C., Octave, J. N., Schneider, Y. J., Trouet, A. &
Crichton, R. R. (1986) Eur. J . Biochem. 155,47-55.
178. Appleton, T. C., Morgan, E. H. & Baker, E. (1971) in The
regulation of erythropoiesis and hemoglobin synthesis
(Travnicek, T. & Neuwirt, J., eds) pp. 310-315, University
Karlova, Praha.
179. Morgan, E. H. (1981) Mol. Aspects Med. 4, 1 - 123.
180. Iacopetta, B. J. & Morgan, E. H. (1984) Biochim. Biophys. Acta
805,211-216.
181. Harding, C., Heuser, J. & Stahl, P. (1983) J . Biol. Chem. 97,
129- 139.
182. McArdle, H. J., Douglas, A. J. & Morgan, E. H. (1985) J. Cell
Physiol122,405 -409.
183. Baker, E., van Bockxmeer, F. M. & Morgan, E. H. (1983) Q. J.
Exp. Physiol. 69, 359 - 372.
184. Baker, E., McArdle, H. J. & Morgan, E. H. (1985) in Proteins
of iron storage and transport (Spik, G., Montreuil, J., Crichton,
R. R. & Mazurier, J., eds) pp. 131 - 142, Elsevier, Amsterdam.
185. Verhoef, N. J. & Noordeloos, P. J. (1977) Clin. Sci. Mol. Med.
52, 87-96.
186. Glass, J., Nunez, M. T. & Robinson, S. H. (1977) Biochem.
Biophys. Res. Commun. 75,226-232.
187. Loh, T. T., Yeung, Y. G. & Yeung, D. (1977) Biochim. Biophys.
Acta471, 118-124.
188. Zaman, Z., Heynen, M. J. & Venvilghen, R. L. (1980) Biochim.
Biophys. Acta 632, 553 - 561.
189. Woodworth, R. C., Brown-Mason, A,, Christensen, T. G., Witt,
D. P. & Comeau, R. D. (1982) Biochemistry 21,4220-4225.
190. Egyed, A. (1984) Br. J . Haematol. 56, 563-570.
191. van der Heul, C., Veldlman, A,, Kroos, M. J. & van Eijk, H. G.
(1984) Int. J . Biochenz. 16, 383-389.
192. Nunez, M. T. & Glass, J. (1985) J . Biol. Chem. 260, 14707-
14711.
193. Laufberger, V. (1973) Bull. SOC.Chim. Biol. 19, 1575-1582.
194. Crichton, R. R. (1973) Struct. Bonding 17,67- 134.
195. Munro, H. N. & Linder, M. C. (1978) Physiol. Rev. 58, 317-396.
196. Harrison, P. M., Hoare, R. J., Hoy, T. G. &Macara, I. G. (1980)
in Iron in biochemistry and medicine, vol. I1 (Jacobs, A. &
Worwood, M., eds) pp. 73 - 114, Academic Press, London.
197. Clegg, G. A., Fitton, J. E., Harrison, P. M. & Treffry, A. (1980)
Prog. Biophys. Mol. Biol. 36, 56 - 86.
198. Theil, E. C. (1983) in Advances in inorganic biochemistry, vol. 5
(Theil, E. C., Eichorn, G. L. & Marzilli, L. G., eds) pp. 1-38,
Elsevier, New York.
199. Mazur, A. & Shorr, E. (1948) J . Biol. Chem. 176,771 -787.
200. Jacobs, A. & Worwood, M. (1975) New Engl. J . Med. 292,951 -
956.
203. Cook, S. F. (1929) J . Biol. Chem. 82, 595-609.
202. Michaelis, L., Coryell, C. D. & Granick, S. (1943) J . Biol. Chem.
148,463 -480.
203. Mann, S., Bannister, J. V. & Williams, R. J. P. (1986) J . Mol.
Biol. 188, 225 - 232.
204. Fischbach, F. A. & Anderegg, J . W. (1965) J . Mol. Biol. 14,
458-473.
205. Harrison, P. M., Fischbach, F. A., Hoy, T. G. & Haggis, G. H.
(1967) Nature (Lond.) 216, 1188-1190.

206. Granick, S. (1942) J . Biol. Chem. 146,451 -461.
207. Towe, K. M. & Bradley, W. F. (1967) J. Colloid Interface Sci.
24,384- 392.
208. Harrison, P. M., Fischbach, F. A., Hoy, T. G. & Haggis, G. H.
(1967) Nature (Lond.) 216, 1188-1190.
209. Massover, W. H. & Cowley, J. M. (1973) Proc. Nut1 Acad. Sci.
USA 70, 3847-3851.
210. Isaacson, M. & Ohtsuki, M. (1980) Scanning Electron Microsc.
1, 72-80.
211. Towe, K. M. (1981) J. Biol. Chem. 256,9377-9378.
212. Chukrov, F. V., Zvyagin, B. B., Gorshkov, A. I., Yermilova, L.
P. & Balashova, V. V. (1973) Znt. Geol. Rev. 16, 1131-1143.
213. Brady, G. W., Kurkjian, C. R., Lyden, E. F. X., Robin, M . B.,
Saltman, P., Spiro, T. & Terzis, A. (1968) Biochemistry 7,
2185 -2192.
214. Heald, S. M., Stearn, E. A., Bunker, B., Holt, E. M. & Holt, S.
L. (1979) J . Am. Chem. Soc. 101,67-73.
215. Theil, E. C., Sayers, D. E. & Brown, M. A. (1979) J . Biol. Chem.
254, 8132-8134.
216. Treffry, A. & Harrison, P. M. (1978) Biochem. J. 171,313-320.
217. Imai, N., Terada, H., Arata, Y . & Fujiwara, S. (1978) Bull.
Chem. Soc. Jpn. 51,2538-2542.
218. Williams, J. M., Danson, D. P. & Janot, C. (1978) Phys. Med.'
Biol. 23, 835-851.
219. Stuhrmann, H. B., Haas, J., Ibel, K., Koch, M. H. J. & Crichton,
R. R. (1976) J . Mol. Biol. 100, 399-413.
220. Massover, W. M. (1978) J . Mol. Biol. 123, 721 -726.
221. Richter, G. W. (1984) Lab. Invest. 50, 26-35.
222. Iancu, T. C. (1983) Mol. Aspects Med. 6, 1-100.
223. Fischbach, F. A., Gregory, D. W., Harrison, P. M., Hoy, T.
G. &Williams, J. M. (1971) J. Ultrastruct. Res. 37, 495-503.
224. Bell, S. H., Weir, M. P., Dickson, D. P. E., Gibson, J. F., Sharp,
G. A. & Peters, T. J. (1984) Biochim. Biophys. Acta 787,227-
236.
225. Hoy, T. G. & Jacobs, A. (1981) Br. J . Haematol. 49, 593-597.
226. Granick, S. & Michaelis, L. (1943) J. Biol. Chem. 147, 91 -97.
227. Harrison, P. M. (1959) J . Mol. Biol. I , 69-80.
228. Leach, B. S., May, M. E. & Fish, W. W. (1976) J . Biol. Chem.
251,3856-3861.
229. Heusterspreute, M. & Crichton, R. R. (1981) FEBS Lett. 129,
322 - 327.
230. Yariv, J., Kalb, A. J., Sperling, R., Bauminger, E. R., Cohen, S.
G. & Ofer, S. (1981) Biochem. J. 197, 171-175.
231. Steifel, E. I. & Watt, G. D. (1979) Nature (Lond.) 279, 81 -83.
232. Chen, M. & Crichton, R. R. (1982) Biochim. Biophys. Acta 707,
1-6.
233. Yariv, J. (1983) Biochem. J . 211, 527-535.
234. Jiudi, L., Jiwen, W., Zepu, Z., Ya, T. & Bei, D. (1980) Sci. Sin.
Engl. Ed. 23, 897 - 904.
235. Niitsu, Y . ,Ishitani, K. & Listowsky, I. (1973) Biochem. Biophyx.
Res. Commun. 55, 1134-1140.
236. Linder, M. C., Moor, J . R. & Munro, H. N. (1974) J . Biol. Chem.
249,7707-7710.
237. Ishitani, K., Niitsu, Y. & Listowsky, I. (1975) J . BioI. Chem.
250,3142-3148.
238. Linder, M. C., Moor, J. R., Munro, H. N. & Morris, H. P.
(1975) Biochim. Biophys. Acta 384,409-421.
239. Bryce, C. F. A., Magnusson, C. G. M. & Crichton, R. R. (1978)
FEBS Lett. 94, 257-262.
240. Drysdale, J. W. (1977) Ciba Found. Symp. 51,41-57.
241. Arosio, P., Adelman, T. G. & Drysdale, J. W. (1978) J. Biol.
Chem. 253,4451 -4458.
242. Arosio, P. (1979) FEBS Lett. 104, 51 - 54.
243. Lavoie, D. J., Ishikawa, K. & Listowsky, I. (1978) Biochemistry
17,5448 - 5454.
244. Watanabe, N. & Drysdale, J. W. (1983) Biochim. Biophys. Acta
743, 98 - 105.
245. Uroshizaki, I., Niitsu, Y., Ishitani, K., Matsuda, M. & Fukuda,
M. (1971) Biochim. Biophys. Acta 243,187-192.
246. Drysdale, J. W. (1970) Biochim. Biophys. Acta 207, 256-258.
247. Bomford, A., Berger, M., Lis, Y .& Williams, R. (1978) Biochem.
Biophys. Res. Commun. 83, 334-341.
505
248. Stefanini, S., Chiancone, E., Arosio, P., Finazzi-Agro, A. &
Antonini, E. (1982) Biochemistry 21, 2293 -2299.
249. Wustefeld, C. & Crichton, R. R. (1982) FEBS Lett. 150, 43-
48.
250. Addison, T. M., Fitton, J. E., Lewis, W. G., May, K. &Harrison,
P. M. (1983) FEBS Lett. 164, 139-144.
251. Crichton, R. R. (1983) in Structure and transport of iron storage
and transportproteins (Uroshizaki, I., Aisen, P., Listowsky, I. &
Drysdale, J. W., eds) pp. 1 - 10, Elsevier, Amsterdam.
252. Liebold, E. A., Aziz, N., Brown, A. J. P. & Munro, H. N. (1984)
J . Biol. Chem. 259,4327-4334.
253. Constanzo, F., Santoro, C., Colantuoni, V., Bensi, G., Raugei,
G., Romano, V. & Cortese, R. (1984) EMBO J. 3, 23 -27.
254. Boyd, D., Vecoli, C., Belcher, D. M., Jain, S. W. & Drysdale, J.
W. (1985)J. Bid. Chem.240,11755-11761.
255. Boyd, D., Jain, S. K., Crampton, J., Barrett, K. J. & Drysdale,
J. (1984) Proc. Nut1 Acad. Sci. U S A 81,4751 -4755.
256. Darner, M. H., Salfeld, J., Will, H., Leibold, E. A,, Vass, J. K. &
Munro, H. N. (1985) Proc. Natl Acad. Sci. USA 82, 3139-
3143.
257. Didsbury, J. R., Theil, E. C., Kaufman, R. E. & Dickey, L. N.
(1986) J . Biol. Chem. 261, 949 -955.
258. Brown, A. J. P., Leibold, E. A. & Munro, H. N . (1983) Proc.
Natl Acad. Sci. USA 80, 1265- 1269.
259. Munro, H. N., Leibold, E. A., Vass, J. K., Aziz, N., Rogers, J.,
Murray, M. & White, K. (1985) in Proteins of iron storage and
transport (Spik, G., Montreuil, J., Crichton, R. R. & Mazurier,
J., eds) pp. 331 -341, Elsevier, Amsterdam.
260. Constanzo, F., Colombo, M., Staempfli, S., Santoro, C.,
Marone, M., Frank, R., Delius, H. &Cortese, R. (1986) Nucleic
Acids Res. 14, 721 -736.
261. Caskey, J. H., Jones, C., Miller, Y . E. & Seligman, P. A. (19113)
Proc. Nut1 Acad. Sci. U S A 80, 482 - 486.
262. Worwood, M., Brook, J. D., Cragg, S. J., Helkuhl, B., Jones, B.
M., Perera, P., Roberts, S. H. & Shaw, D. J. (1985) Hum. Genet.
69, 371 -374.
263. Drysdale, J., Jain, S. K., Boyd, D., Barrett, C., Vecoli, C.,
Belcher, D. M., Beaumont, C., Worwood, M., Lebo, R.,
McGill, J. & Crampton, J. (1985) in Proteins of iron transport
and storage (Spik, G., Montreuil, J., Crichton, R. R. &
Mazurier, J., eds) pp. 343 -347, Elsevier, Amsterdam.
264. Ford, G. C., Harrison, P. M., Rice, D. W., Smith, J . M. A.,
Treffry, A., White, J. L. & Yariv, J. (1984) Phil. Trans. R. Soc.
Lond. B 304,551 -565.
265. Rice, D. W., Ford, G. C., White, J. L., Smith, J. M. A. &
Harrison, P. M. (1983) in Advances in inorganic biochemistry,
vol. 5 (Theil, E. C., Eichorn, G. L. & Marzilli, L. G., eds)
pp. 39-50, Elsevier, New York.
266. Crichton, R. R. (1972) Biochem. J. 130, 35-36P.
267. Rice, D. W., Ford, G. C., White, J. L., Smith, J. M. A. &
Harrison, P. M. (1983) in Structure andfunction ofiron storage
and transportproteins (Uroshizaki, I., Aisen, P., Listowsky, I. &
Drysdale, J. W., eds) pp. 11 - 16, Elsevier, Amsterdam.
268. Otsuka, S., Maruyama, H. & Listowsky, I. (1981) Biochemistry
20, 5226 - 5232.
269. Otsuka, S., Listowsky, I., Niitsu, Y. & Uroshizahki, 1. (1980)
J. Biol. Chem. 255,6234 - 6237.
270. Rice, D. W., Dean, B., Smith, J. M. A,, White, J. L., Ford, G.
C. &Harrison, P. M. (1985) FEBS Lett. 181, 165-168.
271. Massover, W. H. (1979) Biochim. Biophys. Acta 579,169-180.
272. Tsugita, A. & Yariv, J. (1985) Biochem. J . 231, 209-212.
273. Pape, L., Multani, J. S., Saltman, P. & Stitt, C. (1968) Biochemis-
rry 7, 606-612.
274. Spiro, T. G., Allerton, S. E., Renner, J., Terzis, A,, Bils, R. &
Saltman, P. (1966) J . Am. Chem. Soc. 88,2721 - 2726.
275. Reference deleted.
276. Bielig, H. J. & Bayer, E. (1955) Naturwissenschaften 42, 125-
126.
277. Loewus, M. W. & Fineberg. R. A. (1957) Biochim. Biophys. Acta
26,441 -443.
278. Harrison, P. M., Fischbach, F. A,, Hoy, T. G. & Haggis, G. H.
(1967) Nature (Lond.) 216, 1188-1190.
279. Neiderer, W. (1970) Experientia (Basel) 26, 218 -220.
280. Macara, I. G., Hoy, T. G. & Harrison, P. M. (1972) Biochem.
J . 126, 151-162.
281. Bryce, C. F. A. & Crichton, R. R. (1973) Biochern. J . 133, 301 -
309.
282. Macara, I. G., Hoy, T. G. & Harrison, P. M. (1973) Biochem.
J . 135, 343-348.
283. Pbques, E. P., Pbques, A. & Crichton, R. R. (1979) J. Mol.
Catal. 5, 363 - 375.
284. Pbques, E. P., Piques, A. & Crichton, R. R. (1980) Eur. J .
Biochem. 107,447-453.
285. Wauters, M., Michelson, A. M. & Crichton, R. R. (1978) FEBS
Lett. 91, 276-280.
286. Chasteen, N. D. &The& E. C. (1982) J. Biol. Chem. 253,7672-
7677.
287. Crichton, R. R. & Roman, F. (1978) J . Mol. Catal. 4, 75-82.
288. Crichton, R. R. (1981) in The biological chemistry of iron
(Dunford, H. B., Dolphin, D., Raymond, K. N. & Sieker, L.,
eds) pp. 45-61, Reidel, Dordrecht.
289. Wetz, K. & Crichton, R. R. (1976) Eur. J . Biochem. 61, 545-
550.
290. Sayers, D. E., Theil, E. C. & Rennick, F. J. (1983) J . Biol. Chem.
258, 14076- 14079.
291. Chasteen, N. D., Antanaitis, B. C. & Aisen, P. (1985) J . B i d .
Chem. 260,2926 - 2929.
292. Wardeska, J. G., Viglione, B. & Chasteen, N. D. (1986) J . Biol.
Chem. 261,6677-6683.
293. Harrison, P. M., White, J. L., Smith, J. M. A., Farrant, G. W.,
Ford, G. C., Rice, D. W., Addison, J. M. & Treffry, A. (1985)
in Proteins of iron storage and transport (Spik, G., Montreuil,
J., Crichton, R. R. & Mazurier, J., eds) pp. 67-72, Elsevier,
Amsterdam.
294. Hoy, T. G. & Harrison, P. M. (1976) Br. J . Haematol. 33,497-
504.
295. Treffry, A. &Harrison, P. M. (1979) Biochem. J . 181,709-716.
296. Schneider, W. & Erni, I. (1982) in The biochemistry andphysi-
ology of iron (Saltman, P. & Hegenauer, J., eds) pp. 121 - 126,
Elsevier, Amsterdam.
297. Funk, F., Lecrenier, C., Lesuisse, E., Crichton, R. R. &
Schneider, W. (1986) Eur. J . Biochem. 157, 303-309.
298. Mazur, A., Baez, S. & Shorr, E. (1955) J. Bid. Chem. 213, 147-
160.
299. Bielig, H. J. & Bayer, E. (1955) Naturwissenschqften 42, 466-
468.
300. Pape, L., Multani, J. S., Stitt, C. & Saltman, P. (1968) Biochemis-
try 7, 613-616.
301. Dognin, J. & Crichton, R. R. (1975) FEBS Left. 54, 234-236.
302. Tuffano, T. P., Pecoraro, V. L. & Raymond, K. N. (1981)
Biochim. Biophys. Acta 668,420-428.
303. Dognin, J., Girardet, J. L. & Chapron, Y. (1973) Biochim. Bio-
phys. Acta 297,276-284.
304. Crichton, R. R., Roman, F. & Roland, F. (1980) J . Inorg.
Biochem. 13, 305-316.
305. Tidmarsh, G. F., Klebba, P. E. & Rosenberg, L. N. (1983)
J. Inorg. Biochem. 18, 161-268.
306. Bonomi, F. & Pagani, S. (1986) Eur. J . Biochem. 155,295-300.
307. Sirivech, S., Frieden, E. & Osaki, S. (1974) Biochem. J . 143,
311 -315.
308. Crichton, R. R., Roman, F. & Wauters, M. (1975) Biochem.
Soc. Trans. 3, 946-948.
309. Jones, T., Spencer, R. & Walsh, C. (1978) Biochemistry 17,
4011 -4017.
310. Harrison, P. M., Banyard, S. H., Clegg, C. A,, Stammers, D.
K. & Treffry, A. (1978) in Transporf by proteins (Blauer, G. &
Sund, H., eds) pp. 259-275, Walter de Gruyter, Berlin, New
York.
31 1. Funk, F., Lenders, J. P., Crichton, R. R. & Schneider, W. (1985)
Eur. J . Biochem. 152,167-172.
312. Banyard, S. H., Stammers, D. K . & Harrison, P. M. (1978)
Nature (Lond.) 271,282 - 284.
313. Hoare, R. J., Harrison, P. M. & Hoy, T. G. (1975) Nature
(Lond.) 255,653 - 654.
314. Mazur, A., Green, S., Saha, A. & Carleton, A. (1958) J . Clin.
Invest. 53, 1809-1817.
315. Williams, D. M., Lee, G. R. & Cartwright, G. E. (1974) J . Clin.
Invest. 53, 665 - 667.
316. Topham. R. W., Walker, M. C. & Calisch, M. P. (1982) Biochem.
Biophys. Res. Commun. 109,1240- 1246.
317. Thomas, C. E., Morehouse, L. A. & Aust, S. D. (1985) J . Biol.
Chem. 260,3275 -3280.
318. Bienfait, H. F. &Van den Briel, M. L. (1980) Biochim. Biophys.
Acta 631, 507-5510,
319. Wrigglesworth, J. M. & Baum, H. (1980) in Iron in biochemistry
andmedicine, vol. I1 (Jacobs, A. & Worwood, M., eds) pp. 29 -
86, Academic Press, London, New York.
320. Crichton, R. R. (1984) Trends Biochem. Sci. 9, 283-286.
321. Bacon, B. R. & Tavill, A. S. (1984) Sem. Liver Diseases4,lSl-
192.
322. Jacobs, A. (1977) Ciba Found. Symp. 51,91- 100.
323. Jones, M. S. & Jones, 0. T. G. (1969) Biochem. J. 223, 31 - 38.
324. Konopka, K. & Romslo, I. (1980) Eur. J . Biochem. 107, 433-
439.
325. Konopka, K. & Romslo, I. (1981) Eur. J . Biochem. 117, 239-
244.
326. Nilsen, T. & Romslo, I. (1985) Biochim. Biophys. Acta 842,
162-169.
327. Ulvik, R. J. & Romslo, I. (1978) Biochim. Biophys. Acta 541,
251 -262.
328. Ulvik, R. J. & Romslo, I. (1979) Biochim. Biophys. Acta 588,
256-271.
329. Ulvik, R. J., Romslo, I., Roland, F. & Crichton, R. R. (1981)
Biochim. Biophys. Acta 677, 50 - 56.
330. Ulvik, R. J. (1982) Biochim. Biophys. Acta 715,42-51.
331. Flatmark, T. & Tangeras, A. (1977) in Proteins of iron metab-
olism (Brown, E. B., Aisen, P., Fielding, J. & Crichton, R. R.,
eds) pp. 349-358, Grune & Stratton, New York.
332. Tangeras, A,, Flatmark, T., Bickstrom, D. & Ehrenberg, A.
(1980) Biochim. Biophys. Actu 589, 162-175.
333. Tangeras, A. (1985) Biochim. Biophys. Acta. 843, 199-207.
334. Nishisato, T. & Aisen, P. (1982) Br. J . Haematol. 52, 631 -640.
335. Saito, K., Nishisato, T., Grasso, J. A. & Aisen, P. (1986) Br. J .
Haematol. 62, 275 -286.
336. Byers, B. R. & Arceneaux, E. L. (1977) in Microorganisms and
minerals (Weinberg, E. D., ed.) pp. 21 5 - 249, Marcel Decker,
New York.
337. Neilands, J. B. (1979) in Trace metals in health and disease
(Kharasch, N., ed.) pp. 27-41, Raven Press, New York.
338. Spik, G., Fournet, B., Cheron, A., Strecker, G., Montreuil, J.,
Dorland, L. & Vliegenthart, J. F. G. (1979) 5th Int. Symp.
Glycoconjugates, Kiel, pp. 21 - 22.