Molecular size heterogeneity of ferritin in mouse liver

As much as 4% of the total protein in pure liver ferritin from mice with short-term parenteral iron overload produces a minor band migrating anodally to the major (alpha) band of holoferritin with non-denaturing polyacrylamide gel electrophoresis. The components in this minor band and the alpha band have been isolated to purity by preparative electrophoretic fractionation. The protein in the minor band is ferritin, since it contains ferric iron and fulfills defining criteria at the level of biochemistry, immunology and ultrastructure. Native polyacrylamide electrophoresis with pore-size-gradient gels shows that the ferritin molecules in the minor band have a slightly smaller diameter than the holoferritin in the alpha band. Isoelectric focusing reveals that the smaller ferritin has an identical number and range of charge isomers (pI 4.9-5.3) as the larger ferritin, but the relative amount of each size class within some isoferritin bands differs. The smaller ferritin molecules are structurally intact and are made from polypeptide subunits with Mr 18 000; the larger ferritin molecules have subunits with Mr 22 000. The minor species of hepatic ferritin thus has a smaller molecular size because it is made mainly from smaller subunits. No minor electrophoretic band can be detected in liver ferritin obtained from mice with normal iron levels. These results demonstrate that siderosis induces the formation of molecular size polymorphism (macroheterogeneity) in mouse liver ferritin. The new smaller hepatic ferritin could serve to redistribute excess iron into the main storage organs during the early response to iron overload, since it appears to be identical to one of the two types of serum ferritin molecules present in these siderotic mice.     

Influence of heat treatment on rabbit liver ferritin

In order to enhance the stability of beta-galactosidase, we conjugated the enzyme with dextran T-10 (Mr approx. 10 000). The conjugate contained 9-10 mol dextran/mol protein (beta-galactosidase, Mr 68 000), and the specific activity retained after conjugation was 90 +/- 4% (n = 3) of the initial activity. Uptake and degradation of native and conjugated beta-galactosidase in isolated hepatocytes and nonparenchymal liver cells was studied. There was a marked increase in stability against degradation in both cell types when beta-galactosidase was conjugated with Dextran. The degradation of dextran-conjugated enzyme was reduced by 35% in hepatocytes and by 43% in nonparenchymal cells, after 80 and 40 min, respectively, as compared with the free enzyme. However, there was insignificant difference between the uptake of native and conjugated enzyme into the liver cells. Upon intravenous infusion into rats, native and conjugated enzyme were cleared from plasma with only a slight difference in the clearance rate. The observed stability of dextran-conjugated beta-galactosidase towards cellular degradation was in accordance with the in vitro experiments. The conjugate showed marked thermal stability at 50 degrees C and enhanced resistance towards proteolysis by the broad specific protease subtilopeptidase A. This demonstrates that dextran conjugation may be used as a means of stabilizing lysosomal enzymes for therapeutic purposes.     

Purification of a specific repressor of ferritin mRNA translation from rabbit liver

The synthesis of ferritin is regulated at the translation level in coordination with iron availability. Under conditions of low iron, translation of ferritin mRNA is repressed and the majority of ferritin mRNA is non-polysomal. Upon an increase in iron, translation of ferritin mRNA is derepressed resulting in as much as a 50-100-fold increase in the rate of ferritin synthesis. This regulation is mediated at least in part by a specific translational repressor which binds to a conserved sequence, the iron responsive element, located in the 5'-untranslated region of ferritin mRNA. In this communication we report the purification of such a repressor from rabbit liver. This repressor, which we call the "ferritin repressor protein," has an apparent molecular mass of 90 kDa when analyzed by gel filtration chromatography. It inhibits translation of ferritin mRNA in a highly specific fashion when added to a wheat germ lysate programmed with liver poly(A+) mRNA. In addition, it binds specifically to sequences contained within the first 92 nucleotides of ferritin mRNA, most likely the iron responsive element. Analysis of highly purified repressor by sodium dodecyl sulfate-polyacrylamide gel electrophoresis shows that it is composed primarily of a single polypeptide of approximately 90 kDa. Elution of this 90-kDa polypeptide from a sodium dodecyl sulfate gel followed by renaturation and analysis for repressor activity shows that it both binds to the 5'-untranslated region of ferritin mRNA and represses its translation in vitro.     

Iron and phosphorus content of rabbit liver ferritin fractions with different subunit composition

After isolation of subtypes of rabbit liver-ferritin, the phosphorus to iron ratio (P/Fe-ratio) in the samples was found to parallel the shift in subunit composition of the two types of subunits of which ferritins generally consist. No relation was found with the amount of iron per ferritin molecule. The increase of the P/Fe-ratio, in relation to subunit composition, is postulated to be a result of the change of total surface area of the iron microcrystallites inside the ferritin molecule. This surface area depends on the number of nucleation points and thereby may be dependent on the subunit composition.     

Difference of molecular association in two types of curdlan gel

The molecular association in a curdlan gel formed by neutralizing an alkaline solution of curdlan with carbon dioxide was compared with those in gels obtained by heating aqueous suspensions of curdlan at various temperatures.

The neutralized and 60°C-set preparations were soluble in 0·01 sodium hydroxide, whereas preparations set at above 90°C were soluble only in concentrations of sodium hydroxide above 1 . The absorption of Aniline blue or Congo red to the preparations decreased with an increase in the temperature of heat treatment and the adsorption to a gel heated at 120°C for 4 h was about 30% of that for the unheated neutralized gel. Seventy-three per cent of the heated preparation was resistant to treatment with 32% sulfuric acid at 32°C for 30 days, whereas none of the neutralized gel was resistant. An electron micrograph of the resistant part of the curdlan showed that it had a pseudocrystalline form. X-ray studies showed a much higher crystalline structure in the resistant part than in the preparation without heat treatment. The X-ray patterns were almost the same for preparations treated with 32% sulfuric acid or (1 → 3)-β-glucanase.     

A new form of ferritin heterogeneity explained. Isolation and identification of a nineteen-amino-acid-residue fragment from siderosomal ferritin of rat liver.

Ferritin present within siderosomes of iron-loaded rats has a faster anodal mobility than that of cytosolic ferritin from the same rats. A 19-amino-acid-residue peptide was isolated from this fast ferritin and shown to be derived from the C-terminal end of its L-subunit. A 17.3 kDa peptide seen on electrophoresis in denaturing gels of this ferritin accounts for the major portion of the original 182-residue subunit. The two peptides arise from cleavage within the 'insertion region' of the L-subunit sequence that occurs between the D and E helices and lies on the outside of the assembled molecule. This cleavage is present in about 80% of the L-subunits of siderosomal ferritin but nevertheless leaves the molecular structure otherwise intact. It gives rise to an apparent decrease in molecular size, accounting for the faster anodal mobility on native gels. Hence a new form of heterogeneity in ferritin preparations has been explained.     

Protein molecular function influences mutation rates in human genetic diseases with allelic heterogeneity

Molecular epidemiology studies have used the counts of different mutational types like transitions, transversions, etc. to identify putative mutagens, with little reference to gene organization and structure–function of the translated product. Moreover, geographical variation in the mutational spectrum is not limited to the mutational types at the nucleotide level but also have a bearing at the functional level. Here, we developed a novel measure to estimate the rate of spontaneous detrimental mutations called “mutation index” for comparing the mutational spectra consisting of all single base, missense, and non-missense changes. We have analyzed 1609 mutations occurring in 38 exons in 24 populations in three diseases viz. hemophilia B (F9 gene – 420 mutations in 9 populations across 8 exons), hemophilia A (F8 gene – 650, 8 and 26, respectively) and ovarian carcinoma (TP53 gene – 539, 7 and 4, respectively). We considered exons as units of evolution instead of the entire gene and observed feeble differences among populations implying lack of a mutagen-specific effect and the possibility of mutation causing endogenous factors. In all the three genes we observed elevated rates of detrimental mutations in exons encoding regions of significance for the molecular function of the protein. We propose that this can be extended to the entire exome with implications in exon-shuffling and complex human diseases.     

Turnover and tissue uptake of rabbit ferritin from rabbit plasma

Using a two-site immunoradiometric assay for rabbit liver ferritin normal NZW rabbits were found to have very low plasma ferritin concentrations (less than 4 micrograms/l). Purified preparations of rabbit liver and kidney ferritin were labelled with 125I and injected into rabbits. Clearance from plasma was extremely rapid with an initial half-life of 1-2 min as measured by immunoprecipitation of labelled ferritin. The rate of clearance was unaffected by the labelling procedure and by the method of ferritin purification. Autoradiography and organ uptake studies showed that 125I-rabbit liver ferritin was removed mainly by liver reticuloendothelial cells, although on a weight basis, spleen had the greatest radioactivity. These studies indicate that rabbit ferritin released into the circulation is promptly cleared by the RES.     

Caldesmon from rabbit liver: molecular weight and length by analytical ultracentrifugation

Although smooth muscle caldesmon migrates as a 140- to 150-kDa protein during sodium dodecyl sulfate-gel electrophoresis, its molecular mass is around 93 kDa as determined by sedimentation equilibrium (P. Graceffa, C-L. A. Wang, and W. F. Stafford, 1988, J. Biol. Chem. 263, 14,196-14,202). Nonmuscle caldesmon migrates during electrophoresis with a molecular mass close to 77 kDa, about half that of the muscle isoform. However, it is controversial whether the molecular weight of nonmuscle caldesmon is the same or much less than that of the muscle protein. Therefore we have now determined the molecular mass of rabbit liver caldesmon by sedimentation equilibrium and found a value of 66 +/- 2 kDa, a value much smaller than that of muscle caldesmon. This new value of the molecular weight, together with a sedimentation coefficient of 2.49 +/- 0.02 S. yields an apparent length of 53 +/- 2 nm and a diameter of 1.7 nm for the liver protein. We previously estimated a length of 74 nm and a diameter of 1.7 nm for the muscle caldesmon. We have also determined the amino acid composition of liver caldesmon and found it to be similar to that of the muscle protein. In conclusion, muscle and nonmuscle caldesmons appear to have similar overall amino acid composition and tertiary structure with the smaller nonmuscle protein having a correspondingly smaller length. The difference in molecular weight between the two caldesmons is consistent with the nonmuscle protein lacking a central peptide of the muscle isoform, as suggested by E. H. Ball, and T. Kovala, (1988, Biochemistry 27, 6093-6098).     

Induction of VX2 carcinoma in rabbit liver: comparison of two inoculation methods

Direct injection of VX2 cell suspension into the liver is simple and widely used. Implantation of a fragment of VX2 tumour into the liver using a surgical technique has also been developed in the last decade. In this study, we compared these two methods in order to find a better modality for establishing VX2 liver mass. Forty rabbits, each weighing 2.8-3.2 kg, were divided into two groups, 20 rabbits in each. In Group 1, a tumour cell suspension containing 1 x 10(6) cells in a volume of 0.1 ml, was injected slowly into the liver parenchyma using a 27-gauge needle during laparotomy. In Group 2, a 1 mm(3) fragment of VX2 carcinoma was inoculated into the sub-capsule of the left anterior lobe of the liver. In Group 1, three rabbits showed no tumour growth and 10 rabbits showed evidence of leakage and tumour seeding outside of the liver. In Group 2, all but one rabbit showed tumour growth and none showed evidence of tumour seeding. The leakage rates were 50% and 0% for Group 1 and Group 2, respectively. Overall, the success inoculation rate was 35% for Group 1 and 95% for Group 2. In conclusion, to create the VX2 liver tumour model in rabbits, direct implantation of VX2 tumour fragment into the liver achieved better results than injecting cell suspension of VX2 tumour into the liver.     

The distribution of ferritin subunit types in porcine tissues

Ferritin was isolated from porcine heart, liver and spleen. Reversed phase high performance liquid chromatography of the ferritin subunits yielded three chromatographic fractions. The relative proportions of the three chromatographic fractions were different for each tissue ferritin. These results support the model which proposes a combination of (at least) two subunit types as the basis for the existence of isoferritins.