Apoferritin

Clegg, G.A., et al. Helix packing and subunit conformation in horse spleen apoferritin. Nature 288 298-300, 1980. 

Ferritin is another protein that is important in the metabolism of iron. Under normal conditions, it stores iron that can be called upon for use as conditions require. In conditions of excess iron (eg, hemochromatosis), body stores of iron are greatly increased and much more ferritin is present in the tissues, such as the liver and spleen. Ferritin contains approximately 23% iron, and apoferritin (the protein moiety free of iron) has a molecular mass of approximately 440 kDa. Ferritin is composed of 24 subunits of 18.5 kDa, which surround in a micellar form some 3000-4500 ferric atoms. Normally, there is a little ferritin in human plasma. However, in patients with excess iron, the amount of ferritin in plasma is markedly elevated. The amount of ferritin in plasma can be conveniently measured by a sensitive and specific radioimmunoassay and serves as an index of body iron stores. 

Determination of molecular mass of pectic enzymes The molecular mass were determined by gel filtration in a Sepharose CL-6B column (1,8 x 88cm) equilibrated and eluted with Tris-HCl 50 mM, pH 7,5 buffer, plus 100 mM KCl. Fractions (3,3 ml) were collected at a flow rate of 10 ml/h. Molecular mass markers were tyroglobulin (660 kDa) apoferritin (440 kDa) P-amylase (200 kDa) alcohol dehydrogenase (150 kDa) bovine serum albumin (66 kDa) and carbonic anhydrase (29 kDa). Urea-SDS-PAGE (7%) was carried out according to Swank and Munkres [12]. Molecular mass markers were myosin (205 kDa) p-galactosidase (116 kDa) phosphorylase b (97 kDa) bovine serum albumin (66 kDa), ovalbumin (45 kDa) and carbonic anhydrase (29 kDa). 

Figure 3. (A) Determination of molecular mass of pectic enzymes by gel filtration in Sepharose 6B. Molecular mass markers - tyroglobulin, 2- apoferritin, 3- p-amylase, 4-alcohol dehydrogenase, 5- bovine serum albumin, 6- carbonic anhydrase. (B) SDS-PAGE of pectolytic activities. Molecular mass markers 1- myosin, 2- p-galactosidase, 3- phosphorylase b, 4- bovine serum albumin, 5- ovalbumin, 6- carbonic anhydrase.

Ferritins have been found in a wide range of species, and sequence data - some, as in the first ever sequence of horse spleen apoferritin (Heusterspreute and Crichton, 1981) determined by direct methods, but many now by DNa sequencing 1, have been deposited for more than 70 ferritins. They vary in length from 154-185 residues per subunit. Some ferritins have N-terminal extensions which lie on the outside of the assembled shell and target the ferritin to a specific destination such as plastids in plants and yolk sac in snails (Andrews etah, 1992 Lobreaux etah, 1992). For example, pea ferritin is synthesized with an N-terminal extension of 75 residues, which is missing from the mature protein. The first part of this extension is a chloroplast-targetting sequence of 47 residues, which is lost on entry into the plastid. The second part, an extension peptide, is lost prior to assembly of the... 

Horse-spleen apoferritin crystallizes in a face-centred, close-packed, cubic arrangement, in the space group F432, with molecules at the 432 symmetry points of the crystal lattice (Harrison, 1959). This publication was the logical extension of the DPhil thesis of the Oxford chemist Pauline M. Cowan (as she was before her marriage to Roy Harrison), and represented the first publication in what was to be a long and distinguished series of contributions on ferritin from the undisputed Iron Lady of iron metabolism. ... 

The crystallization of horse spleen apoferritin was in fact a fortuitous coincidence, because, as we mentioned earlier, attempts to crystallize horse-liver ferritin were not successful, whereas the iron-rich ferritin from horse spleen could be crystallized (Laufberger, 1937). This was certainly related to the relatively high content of H subunits (average composition L12H12) in horse liver (something that was only discovered 50 years later). It has generally proved very difficult if not impossible to crystallize heteropolymers, and the best results in crystallographic terms have been obtained with recombinant homopolymers. As will be discussed later in this chapter,... 

The arrangement of the 24 subunits of the apoferritin molecule in their 432 symmetry viewed down a fourfold axis is presented in Figure 6.3. Also included in Figure 6.3 is a labelling scheme of symmetry related subunits and a representation of the subunit as a ribbon diagram of the -carbon backbone. Of the 174 amino-acid residues of the L-chain 140 (80 %) are found in five a-helices. Each of the 24 subunits consists... 

When pea seed apoferritin is reconstituted in vitro in the absence of phosphate, the reconstituted mineral core consists of crystalline ferrihydrite (Rohrer et ah,1990 Wade et ah, 1993 Waldo et ah, 1995). Conversely, horse spleen ferritin reconstituted in the presence of phosphate produces an amorphous core (Rohrer et ah,1990 St. Pierre et ah, 1996)... 

Several binding sites for Tb3+ or Cd2+ ions have been identified in the interior of the apoferritin protein shell, some of which may be iron-binding sites (Harrison et ai, 1989 Granier et ah, 1998). In HoSF and HoLF, two sites were identified on the inner surface of the B helix at the subunit dimer interface (Figure 6.15, Plate 11) which bind two Cd2+ ions. One involves Glu-57 and Glu-60 as ligands and the other Glu-61 and Glu-64 (Granier et al., 1998). In H-chain ferritins the first pair of Glu-57 and Glu-60 are both replaced by His and only a single Tb3+ is found bound to Glu-61 and Glu-64 (Lawson et al, 1991). 

We now consider how the biomineralization chamber which is constituted by the interior of the apoferritin protein shell influences the growth of the core. Once sufficient core has been developed (>100 Fe atoms), Fe(II) oxidation and hydrolysis can proceed (Yang et ah, 1998) on the mineral surface of the growing core (equation 8) ... 

Iron oxidized by ferritin must be at or near the outer surface of the apoferritin molecule, since iron appears to be exchanged between ferritin molecules, as shown by Mossbauer spectroscopy (Bauminger et ah, 1991a,b) and by the observation that iron oxidized by ferritin can be taken up directly by apotransferrin (Bakker and Boyer, 1986 Jin and Crichton, 1987). 

The underlying chemistry of iron biomineralization has been discussed in Chapter 1, and in this present section we discuss the way in which the apoferritin protein... 

It is unfortunately the case that when we incubate apoferritin with a certain number of iron atoms (for example as ferrous ammonium sulfate), the product, after elimination of non-protein-bound iron, does not have a homogeneous distribution of iron molecules which were able to (i) take up iron rapidly through the three fold channels, (ii) quickly transfer it and form a diiron centre on a ferroxidase site, and (iii) to transfer the iron inward to a nucleation site, where (iv) it will begin to catalyse iron oxidation on the surface of the growing crystallite, will accumulate iron much more rapidly, and in much greater amounts than molecules in which steps (i), (ii) and (iii) are slower, for whatever reasons (perhaps most importantly subunit composition, and the disposition of subunits of the two types H and L, one with regard to the other). This polydispersity makes the analysis of the process of iron uptake extremely difficult. 

Chasteen, N.D. and Theil, E.C. 1982. Iron binding by horse spleen apoferritin a vanadyl(IV) EPR spin probe study. Journal of Biological Chemistry 257 7672-7677. 

We assume that all substances involved in ferritin iron deposition (Fe2+, Fe3+, 02) need to gain access to the interior of the apoferritin protein shell. The most likely pathway is via the three-fold channels, which would involve passing through the 12 A long channel, and then traverse a further distance of 8 A along a hydrophilic pathway from the inside of the... 

We now consider how the biomineralization chamber which is constituted by the interior of the apoferritin protein shell influences the growth of the core. Once sufficient core has... 

Toussaint, L., Bertrand, L., Hue, L., Crichton, R.R. and Declercq, J.-P. (2006) High-resolution X-ray structures of human apoferritin H-chain mutants correlated with their activity and metal-binding sites, J. Mol. Biol., 365, 440-452. 

Fig. 6.29 S(Q,t)/S(Q) measured at the IN15 on a 156 mg/mL apoferritin solution with 0.01 mM salt. Despite the paracrystalline order no permanent restriction of motion is present as indicated by the virtually full decay of the relaxation curve (Q=0.09 A ). (Reprinted with permission from [333]. Copyright 2003 Elsevier)...

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