Structural studies on the iron storage protein, apoferritin

  • Margaret Paterson

    Student thesis: Doctoral Thesis

    Abstract

    Horse spleen apoferritin consists of 24-subunits symmetraically arranged around a central iron-containing cavity. It was originally thought that the 24 subunits were identical, however evidence based on SDS-PAGE and isoelectrofocusing led Drysdale to propose a model with two distinct subunits, the L subunit (m.wt. 19,000) and H subunit (m.wt 21,000) which are combined in different proportions to give various 24-mers (isoferritins). This model has been disputed by several workers.

    When horse spleen apoferritin is electrophoresed by SDS-PAGE it resolves into several bands. The origin of these bands were studied. Bands were visible with molecular weights above and below that of the subunit. Using a 2-dimensional technique several of these band could be attributed to inter-molecular disulphide bonds forming dimers and trimers and an intra-molecular disulphide bond giving an apparently smaller subunit band (m.wt. 15,000). Other smaller molecular weight bands have been re-assessed by SDS-PAGE to have molecular weights of 11,000 (peptide B) and J+, 500 (peptide C). Aminoacid analysis indicates peptide C arises from the C-terminal end of the molecule. Sequencing of this peptide gave the following sequence:

    leu-thr-leu-lys-aromatic-trp

    which corresponds to the last six amino acids of the published sequence. Peptide C could therefore extend beyond this sequence and one explanation of this could be that the H subunit is a precursor of the L subunit.

    A glycoprotein affinity column showed that glycosylated subunits did occur but the extent of glycosylation was insufficient to affect the subunit molecular weight on SDS-PAGE but may be sufficient to influence isoelectro-focusing profiles.

    Solid-phase sequencing studies carried out on isolated L and H-rich subunits showed that both were resistent to sequencing from the N-terminal end. This implies that H as well as L subunits are H-blocked.

    Chemical modifications, cleavage and spectrophotometric studies indicated the presence of 1 tryptophan, 5-6 tyrosine and 2 cysteine residues which corresponds well with the published sequence.

    Tissue culture experiments carried out on Morris hepatoma cells showed ferritin synthesis could be increased up to 5-fold greater than control cells by introducing iron into the culture medium. Initial studies were also carried out to determine if either subunit (L or H) was preferentially synthesized during iron loading. Studies of this type may provide information on whether two subunits do exists and if so their function within the molecule.
    Date of Award1984
    LanguageEnglish
    Awarding Institution
    • Council for National Academic Awards

    Cite this

    Structural studies on the iron storage protein, apoferritin
    Paterson, M. (Author). 1984

    Student thesis: Doctoral Thesis