Mouse hepatoma and liver ferritins. Comparative structural studies.

Pure ferritin from male mouse liver produces a single band of monomers (RF = 0.199) with electrophoresis in polyacrylamide gels at pH 9.0. The five sub-bands within this monomeric band appear to represent charge isomers having the same molecular size. Ferritin from BH3 transplantable mouse hepatoma shows two overlapping bands of monomers (RFA = 0.208 and RFB = 0.240); further electrophoretic studies show that these bands represent two subpopulations of molecules differing both in charge and size. Sub-bands are not found in this hepatoma ferritin. The larger tumor ferritin reaches the same end migration position as all liver isoferritins on gradient gels, signifying a very similar or identical molecular size; however, the absence of sub-bands indicates that this hepatoma ferritin differs in charge from the homologous liver proteins. Liver and hepatoma ferritins both produce a single prominent subunit band corresponding to nominal molecular weights of 22 250 and 21 700, with polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate and dithiothreitol. With electrophoresis on polyacrylamide gradient slabs containing sodium dodecyl sulfate and dithiothreitol, both liver and hepatoma ferritins now reveal two subunits bands situated at identical positions. The polypeptides of these two closely spaced bands have a nominal molecular weight difference of less than 1000. Neither the hepatoma nor the liver seems to produce the ferritins found in the other tissue. Nevertheless, all these ferritins are composed of the same two types of subunits, albeit in different relative amounts. Observed distinctions in the ferritins from these normal or neoplastic cells must reflect differences in assembly and processing, as well as in the regulated expression of the same ferritin genes.     

Siderosomal ferritin. The missing link between ferritin and haemosiderin?

A minor electrophoretically fast component was found in ferritin from iron-loaded rat liver in addition to a major electrophoretically slow ferritin similar to that observed in control rats. The electrophoretically fast ferritin showed immunological identity with the slow component, but on electrophoresis in SDS it gave a peptide of 17.3 kDa, in contrast with the electrophoretically slow ferritin, which gave a major band corresponding to the L-subunit (20.7 kDa). Thus the electrophoretically fast ferritin resembles that reported by Massover [(1985) Biochim. Biophys. Acta 829, 377-386] in livers of mice with short-term parenteral iron overload. The electrophoretically fast ferritin had a lower iron content (2000 Fe atoms/molecule) than the electrophoretically slow ferritin (3000 Fe atoms/molecule). Removal and re-incorporation of iron was possible without effect on the electrophoretic mobility of either ferritin species. On subcellular fractionation the electrophoretically fast ferritin was enriched in pellet fractions and was the sole soluble ferritin isolated from iron-laden secondary lysosomes (siderosomes). The amount and relative proportion of the electrophoretically fast species increased with iron loading. Haemosiderin isolated from siderosomes was found to contain a peptide reactive to anti-ferritin serum and corresponding to the 17.3 kDa peptide of the electrophoretically fast ferritin species. Unlike the electrophoretically slow ferritin, the electrophoretically fast ferritin did not become significantly radioactive in a 1 h biosynthetic labelling experiment. We conclude that the minor ferritin is not, as has been suggested for mouse liver ferritin, 'a completely new species of smaller holoferritin that represents a shift in the ferritin phenotype' in response to siderosis, but a precursor of haemosiderin, in agreement with the proposal by Richter [(1984) Lab. Invest. 50, 26-35] concerning siderosomal ferritin.     

Protein heterogeneity in rabbit liver ferritin: two types of molecular dimers

Native pore-gradient polyacrylamide gel electrophoresis of rabbit liver ferritin reveals the usual single band of molecular monomers, but shows two bands at the position of molecular dimers. The proteins in these three bands were purified by excision from preparative slab gels. All three bands (1) contain considerable amounts of iron-rich ferritin when examined by electron microscopy, (2) show complete identity when reacted with anti-rabbit-ferritin antibodies, and (3) have similar amounts of H-type and L-type ferritin subunits with denaturing polyacrylamide gel electrophoresis. These results establish that there are two classes of ferritin molecular dimers. The larger dimer band uniquely also contains a polypeptide with Mr = 170,000. This unusual type of ferritin heterogeneity seems to be due to the presence of a non-ferritin protein associated only with one class of dimers.     

Size and charge heterogeneity of rat tissue ferritins.

Ferritins purified from horse spleen and from rat liver, kidney, heart and hepatoma were analyzed by quantitative polyacrylamide gel electrophoresis. From the migration characteristics of these ferritins at several gel concentrations, Ferguson plots were constructed and the molecular sizes and charges (apparent valences) together with their statistical variability were obtained by applying Rodbard computer programs to the data. Finally, ellipses were drawn describing the 95% confidence limits of these data for size and charge and were used to identify those ferritins that differed in size and/or charge. By these criteria, many of the tissue ferritins were differentiated from one another in terms of their molecular size and/or charge. Among the various tissue ferritin monomers, the molecular sizes were essentially similar (420 000-490 000) except for the two heart ferritins which were larger (530 000 and 626 000, respectively). However, the estimated charges on rat liver, kidney and hepatoma monomers (30-38 net protons per molecule) differed from that of spleen monomer (51 net protons per molecule) while the larger rat heart ferritin also had a greater charge (83 net protons) than the smaller (40 net protons). Apoferritins prepared chemically by removal of iron from the holoferritins had migration properties indistinguishable from the parent holoferritins. The migration properties of minor (dimeric) ferritin bands on the gels were compared with those of the monomer bands. The molecular sizes of the minor bands were larger than those of the major bands, and were not inconsistent with a doubling in size. However, charge differences varied, being either similar for major and minor forms (spleen ferritin), approximately twice for the minor form (rat hepatoma ferritin) or five times greater for the minor form (rat liver ferritin). These differences in behavior were confirmed by using minimally sieving gels, on which the major bands of horse spleen ferritin failed to separate whereas those of rat liver ferritin were readily separable. It is concluded that dimers of ferritins from different tissues may associate in different ways.     

Secreted ferritin subunits are of two kinds in insects molecular cloning of cDNAs encoding two major subunits of secreted ferritin from Calpodes ethlius

In insects, holoferritin is easily visible in the vacuolar system of tissues that filter the hemolymph and, at least in Lepidoptera, is abundant in the hemolymph. Sequences reported for insect secreted ferritins from Lepidoptera and Diptera have high sequence diversity. We examined the nature of this diversity for the first time by analyzing sequences of cDNAs encoding two ferritin subunits from one species, Calpodes ethlius (Lepidoptera, Hesperiidae). We found that insect secreted ferritin subunits are of two types with little resemblance to each other. Ferritin was isolated from iron loaded hemolymph of C. ethlius fifth instar larvae by differential centrifugation. The N-terminal amino acid sequences for the nonglycosylated subunit with Mr 24,000 (S) and the largest glycosylated subunit with Mr 31,000 (G) were determined. The N-termini of the two subunits were different and were used to construct degenerate PCR primers. The same cDNA products were amplified from cDNA libraries from the midgut which secretes holoferritin and from the fat body which secretes iron-poor apoferritin. The G subunit most closely resembles the glycosylated ferritin subunit from Manduca sexta and the S subunit resembles the Drosophila small subunit. The S and G subunits from Calpodes were dissimilar and distinct from the cytosolic ferritins of vertebrates and invertebrates. Additional sequences were obtained by 5' and 3' RACE from separate fat body and midgut RACE libraries. cDNAs encoding both subunits had a consensus iron responsive element (IRE) in a conserved cap-distal location of their 5' UTR. An integrin-binding RGD motif found in the G subunit and conserved in Manduca may facilitate iron uptake through a calreticulin (mobilferrin)/integrin pathway. Calpodes and other insect ferritins have conserved cysteine residues to which fatty acids can be linked. Dynamic acylation of ferritin may slow but not prevent its passage out of the ER.     

The characterization of ferritin in an insect

In mammals, the iron storage protein ferritin is predominantly synthesized on free polysomes and accumulates in the cytosol but some is secreted and circulates in the blood as serum ferritin. In insect tissues, on the other hand, iron-containing holoferritin accumulates in the vacuolar system and can be secreted through the Golgi complex. The midgut can secrete it to the gut lumen and other tissues to the hemolymph.Ferritin was isolated from the midgut and hemolymph of fifth instar larvae of Calpodes ethlius, Lepidoptera, Hesperiidae. This holoferritin is stable to heat (75°C) or in the presence of SDS, proteinase K, or urea, has an Mr above 600,000, contains iron and resembles mammalian ferritins in appearance by electron microscopy. Calpodes ferritin is a glycoprotein having N-linked high-mannose oligosaccharides. It is not antigenically related to horse ferritin but is related to that from Manduca sexta, Lepidoptera, Sphingidae. In its native form, Calpodes ferritin has only 3 isoforms with a pI 6.5–7 suggesting a more uniform subunit composition than that in vertebrates. It has two principle subunits, with relative Mrs of 24,000 (L) and 31,000 (G) and two minor subunits with Mrs of 26,000 and 28,000 all of which cross-react with antibody to Manduca ferritin. The 24 kDa subunit is the only one that is not glycosylated. Iron injections induce an increase in the proportion of the 24 kDa subunit. We conclude that Calpodes has ferritin and that it is glycosylated like mammalian serum ferritin.     

Stoichiometry of Fe(II) oxidation during ceruloplasmin-catalyzed loading of ferritin.

Ceruloplasmin catalyzed the incorporation of iron into apoferritin with a stoichiometry of 3.8 Fe(II)/O2. This value remained the same when ferritin containing varying amounts of iron was used. Contrary to the "crystal growth" model for ferritin formation, no iron incorporation into holoferritin was observed in the absence of ceruloplasmin. Fe(II)/O2 ratios close to 2 were obtained for iron incorporation into apo- and holoferritin in Hepes buffer, in the absence of ceruloplasmin, indicating the formation of reduced oxygen species. Sequential loading of ferritin in this buffer resulted in increasing oxidation of the protein as measured by carbonyl formation. Sequential loading of ferritin using ceruloplasmin did not result in protein oxidation and a maximum of about 2300 atoms of iron were incorporated into rat liver ferritin. This corresponded to the maximum amount of iron found in rat liver ferritin in vivo after injection with iron. These results provide evidence for ceruloplasmin as an effective catalyst for the incorporation of iron into both apo- and holoferritin. The possibility that these findings may have physiological significance is discussed.     

Prenatal and postnatal changes in the content and species of ferritin in rat liver

The iron and ferritin content of rat liver and the species of ferritin present were examined from 4 days before to 3 weeks after birth. 1. Total iron and ferritin iron accumulated rapidly during the last days of gestation and from the second postnatal day underwent a steady depletion. 2. The amount of iron deposited before birth in the liver of each pup varied inversely with litter size and could be increased moderately by injection of iron into the mother before mating. 3. Intraperitoneal injection of iron 1 day after birth doubled the concentration of total iron, ferritin iron and ferritin protein in the liver over the next 24h, but at 3 weeks after birth it raised the very low concentrations of iron and ferritin severalfold. 4. As shown by electrophoretic migration, ferritin and dissociated ferritin subunits prepared from the livers of rats from 4 days before to 3 weeks after birth differed from those of adult liver ferritin and were indistinguishable from those of adult kidney and spleen ferritin. Treatment with iron at 3 weeks of age induced formation of a ferritin with electrophoretic properties resembling those of adult liver. It is concluded that iron given at this stage of development may activate the genetic cistron for adult liver ferritin.     

Proteomic analysis of hepatic iron overload in mice suggests dysregulation of urea cycle, impairment of fatty acid oxidation, and changes in the methylation cycle

Liver iron overload can be found in hereditary hemochromatosis, chronic liver diseases such as alcoholic liver disease, and chronic viral hepatitis or secondary to repeated blood transfusions. The excess iron promotes liver damage, including fibrosis, cirrhosis, and hepatocellular carcinoma. Despite significant research effort, we remain largely ignorant of the cellular consequences of liver iron overload and the cellular processes that result in the observed pathological changes. In addition, the variability in outcome and the compensatory response that likely modulates the effect of increased iron levels are not understood. To provide insight into these critical questions, we undertook a study to determine the consequences of iron overload on protein levels in liver using a proteomic approach. Using two-dimensional polyacrylamide gel electrophoresis (2D-PAGE) combined with matrix-assisted laser desorption ionization mass spectrometry (MALDI-MS), we studied hepatic iron overload induced by carbonyl iron-rich diet in mice and identified 30 liver proteins whose quantity changes in condition of excess liver iron. Among the identified proteins were enzymes involved in several important metabolic pathways, namely the urea cycle, fatty acid oxidation, and the methylation cycle. This pattern of changes likely reflects compensatory and pathological changes associated with liver iron overload and provides a window into these processes.     

Ferritin in liver, plasma and bile of the iron-loaded rat

Rats were loaded with iron. With overload, up to a 10-fold increase of the iron and ferritin protein content of the livers was measured. The plasma ferritin concentration increased gradually with the ferritin concentration in the liver. The ferritin concentration in the bile increased also and was in the same range as in the plasma. The ratio plasma ferritin concentration to bile ferritin concentration in individual rats decreased in the case of considerable iron overload. After intravenous injection of liver ferritin, less than 2% of the ferritin concentration that disappeared from the blood was found to be in the bile. Isoelectric focussing revealed that the microheterogeneity of liver and bile ferritin were identical, but slightly different from plasma ferritin. These results indicate that ferritin was not solely leaking from the plasma to the bile. Together with ferritin, iron accumulated in the bile. The iron content of the bile ferritin was in the same range as in fully iron-loaded liver ferritin. It is likely that ferritin in the bile is excreted by the liver and consists of normal iron-loaded liver ferritin molecules. In all circumstances, the amount of iron in the bile was much higher than could be accounted for by transport by the bile ferritin. The ferritin protein to iron ratio in the bile was 0.1-1.2, which was in the same range as was measured in isolated lysosomal fractions of the liver. Those results agree with the supposition that ferritin and iron in the bile are excreted by the liver though lysosomal exocytosis.     

MITOCHONDRIAL AND CYTOPLASMIC RIBOSOMES FROM TETRAHYMENA PYRIFORMIS : Correlative Analysis by Gel Electrophoresis and Electron Microscopy

Mitochondrial and cytoplasmic ribosomes from Tetrahymena pyriformis have been isolated and studied by the techniques of polyacrylamide gel electrophoresis and electron microscopy used in conjunction. Although the two ribosome types show the same coefficient of sedimentation (80S) in sucrose gradients, they can be distinguished by gel electrophoresis: mitoribosomes migrate in a single band, considerably slower than the cytoribosome band. Electron microscope observations of negatively stained cytoribosomes show typical rounded or triangular profiles, about 275 x 230 Å; mitoribosome profiles are much larger and clearly elongate, about 370 x 240 Å. An electron-opaque spot delimits two nearly equal size subunits. In mixtures of mito- and cytoribosomes, each type can be recognized by its characteristic electrophoretic mobility and by its distinctive fine structure. Cytoribosomal 60S and 40S subunits each produce a distinct electrophoretic band. On the contrary, neither electrophoretic analysis, using a variety of conditions, nor electron microscopy is able to discern two different subunit types in the single 55S mitoribosomal subunit peak. Electrophoretic analysis of RNA shows that both ribosomal RNA species are present in the mitoribosomal subunit fraction. These results establish that mitoribosomes from T. pyriformis dissociate into two subunits endowed with the same sedimentation coefficient, the same electrophoretic mobility, and a similar morphology.     

Purification of RNAase II by preparative polyacrylamide gel electrophoresis.

Purification of RNAase II to electrophoretic homogeneity is described. The exonuclease is activated by K+ and Mg2+ and hydrolyses poly(A) to 5'-AMP, exclusively as described by Nossal and Singer (1968, J. Biol. Chem. 243, 913--922). To separate RNAase II from ribosomes, DEAE-cellulose chromatography was used. Two additional chromatographic steps give a preparation that yields 10 bands after analytical polyacrylamide gel electrophoresis. Preparative polyacrylamide gel electrophoresis resulted in a final preparation which on analytical polyacrylamide gels gives a single band. A molecular weight of 76 000 +/- 4000 was obtained from Sephadex G-200 chromatography, with three bands from sodium dodecyl sulfate (SDS) denaturation and SDS gel electrophoresis. The subunits have a molecular weight of 40 000 +/- 2000, 33 000 +/- 2000, and 26 000 +/- 1000. The enzyme thus appears to consist of three dissimilar subunits.     

Characteristics of structure, composition, mass spectra, and iron release from the ferritin of shark liver (Sphyrna zygaena)

The ferritin consists of a protein shell constructed of 24 subunits and an iron core. The liver ferritin of Sphyrna zygaena (SZLF) purified by column chromatography is a protein composed of eight ferritins containing varying iron numbers ranging from 400+/-20 Fe3+/SZLF to 1890+/-20 Fe3+/SZLF within the protein shell. Nature SZLF (SZLFN) consisting of holoSZLF and SZLF with unsaturated iron (SZLFUI) to have been purified with polyacrylamide gel electrophoresis (PAGE) exhibited five ferritin bands with different pI values ranging from 4.0 to 7.0 in the gel slab of isoelectric focusing (IEF). HoloSZLF purified by PAGE (SZLFE) not only had 1890+/-20 Fe3+/SZLFE but also showed an identical size of iron core observed by transmission electron microscopy (TEM). Molecular weight of approximately 21 kDa for SZLFE subunit was determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Four peaks of molecular ions at mass/charge (m/z) ratios of 10611.07, 21066.52, 41993.16, and 63555.64 that come from the SZLFE were determined by matrix-assisted laser desorption ionization/time-of-flight mass spectrometry (MALDI-TOF MS), which were identified as molecular ions of the ferritin subunit (M+) and its polymers, namely, [M]2+, [M]+, [2M]+, and [3M]+, respectively. Both SZLFE and a crude extract from shark liver of S. zygaena showed similar kinetic characteristics of complete iron release with biphasic behavior. In addition, a combined technique of visible spectrometry and column chromatography was used for studying ratio of phosphate to Fe3+ within the SZLFE core. Interestingly, this ratio maintained invariable even after the iron release, which differed from that of other mammal ferritins.     

Isolation and partial amino acid sequence of three subunit species of porcine spleen ferritin: evidence of multiple H subunits

A partial amino acid sequence for three different subunits of the iron storage protein, ferritin, has been determined. Ferritin (Mr approximately 480,000) was isolated from porcine spleen and dissociated into its component subunits (Mr approximately 20,000). The subunits, in turn, were separated into three fractions by reversed-phase HPLC. The fractions appeared to be of equal size by sedimentation velocity, sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and size-exclusion chromatography in 6 M guanidinium chloride. All three fractions were shown to be monomeric and to have no covalently attached carbohydrate (J. F. Collawn et al. (1984) Arch. Biochem. Biophys. 233, 260-266). Determination of the amino acid sequence of the C-terminal 70-80 residues from each of the fractions demonstrated three different sequences. Comparison with human liver H and L subunit sequences indicates that two of the porcine ferritin subunits are H-type subunits and one is an L-type subunit. Application of the Chou-Fasman algorithm on the three partial sequences suggests that these respective regions from each of the three subunits would probably adopt the same conformation.     

Uterine myometrial ferritin and blood plasma transferrin as natural modulators of Ca-calmodulin-independent cyclic nucleotide phosphodiesterase from cow uterine myometrium

The iron storage protein ferritin was purified to electrophoretic homogeneity from cow uterine myometrium. Its Mr did not exceed 440, 000 and H-chains predominated in the subunit composition; the iron saturation was 43 iron ions per protein molecule. The uterine myometrial ferritin was a potent natural modulator of Ca-calmodulin-independent phosphodiesterase (Ca-CM-independent PDE, EC 3.1.4.17) isolated from the same tissue. Addition of iron-poor ferritin from uterine myometrium and iron-reach liver ferritin caused three- and two-fold inhibition of the enzyme activity, respectively. The iron transport protein transferrin in iron-saturated and iron-depleted forms can also inhibit Ca-CM-independent PDE activity by two-fold. In both cases, the degree of saturation with iron was not crucial for the inhibitory effects of these proteins on the enzyme activity. These data suggest that iron homeostasis proteins can modulate the cyclic nucleotide level in non-nervous tissue via interaction with enzymes involved in cyclic nucleotide hydrolysis.     

Phosphorylation of the insulin receptor by casein kinase I

Insulin receptor was examined as a substrate for the multipotential protein kinase casein kinase I. Casein kinase I phosphorylated partially purified insulin receptor from human placenta as shown by immunoprecipitation of the complex with antiserum to the insulin receptor. Analysis of the phosphorylated complex by polyacrylamide gel electrophoresis under nonreducing conditions showed a major phosphorylated band at the position of the alpha 2 beta 2 complex. When the phosphorylated receptor was analyzed on polyacrylamide gels under reducing conditions, two phosphorylated bands, Mr 95,000 and Mr 135,000, were observed which corresponded to the alpha and beta subunits. The majority of the phosphate was associated with the beta subunit with minor phosphorylation of the alpha subunit. Phosphoamino acid analysis revealed that casein kinase I phosphorylated only seryl residues. The autophosphorylated alpha 2 beta 2 receptor purified by affinity chromatography on immobilized O-phosphotyrosyl binding antibody was also a substrate for casein kinase I. Reduction of the phosphorylated alpha 2 beta 2 receptor indicated that casein kinase I incorporated phosphate into seryl residues only in the beta subunit.     

Isolation and characterization of Phycomyces blakesleeanus ferritin.

Ferritin was isolated from the fungus Phycomyces blakesleeanus and compared biochemically and immunologically with horse spleen ferritin. Phycomyces and horse spleen ferritins were shown to exhibit similar electrophoretic patterns on polyacrylamide gels. Both preparations yielded an identical single band on sodium dodecyl sulfate-containing polyacrylamide gels. Tryptic digests of Phycomyces ferritin yielded 17 ninhydrin-positive spots as compared to 26 for horse spleen ferritin tryptic digests. Phycomyces ferritin was immunologically unrelated to horse spleen ferritin.     

Inactive hepatic lipase in rat plasma

Hepatic lipase activity is detectable in liver but also in adrenal glands, ovaries, and plasma. The subunit size of hepatic lipase in liver, adrenal glands, and nonheparin plasma was compared. Hepatic lipase in liver and adrenal glands appeared as a 55 kDa band. In liver, a faint band of lower size was also detected. In nonheparin plasma, hepatic lipase appeared as a doublet of 57 kDa and 59 kDa. When activity/mass ratio was calculated, similar values were obtained for liver and adrenal glands. In plasma this value was much lower. After heparin administration in vivo, hepatic lipase activity in plasma increased nearly 100-fold with appearance of an additional 55 kDa band in postheparin plasma. This band coeluted with activity after preparative polyacrylamide gel electrophoresis. Differences in size persisted after digestion with peptide-N-glycosidase F. A progressive increase in 57 kDa and 59 kDa in postheparin plasma followed disappearance of the 55 kDa band, suggesting that these larger bands originate from the smaller form. In plasma, both smaller and larger forms were associated with HDL, but not with LDL or VLDL. We conclude that rat plasma contains a larger form of hepatic lipase that is inactive in in vitro assay.