Structural and immunological relationships of isoferritins in normal and malignant cells.

Ferritins from normal adult human liver and heart were compared with ferritins from a lung carcinoma metastatic to liver and from HeLa cells on the basis of their isoferritin profiles, subunit composition, and immunological relationships. Each ferritin preparation gave different isoferritin profiles, but several contained common isoferritins. All of the tumor isoferritins had counterparts in the normal tissues. All ferritins contained similar subunits but in different proportions. Qualitative differences were demonstrable in some ferritins with antibodies to different tissue ferritins. These differences correlated with the subunit composition of the ferritins. By appropriate absorption, an antibody population was obtained that was apparently specific for one subunit type. Heart ferritin gave lines of apparent identity with the tumor ferritins with these antibodies. It is concluded that tumor ferritins are not tumor-specific antigens but correspond to isoferritins in normal adult heart.

The properties of Corbicula sandai apoferritin.

Corbicula sandai apoferritin possesses physical properties different from apoferritins of other species. The native molecular weight was estimated from its s020,w of 18.7 S to be about 503 000. Empirical molecular weight estimation methods in denaturing solvents yielded a molecular weight estimate for the constituent polypeptide chain of 23 000. The circular dichroic spectrum of C. sandai apoferritin was significantly different from other apoferritins and it was immunologically unreactive with rabbit anti-human ferritin antisera.

Primary structure of rat liver apoferritin. The amino end.

Rat liver apoferritin is known to have a blocked amino end. From a pronase digest of rat liver apoferritin we have isolated and purified by ion-exchange chromatography the blocked N-terminal tripeptide. Its sequence and the nature of the blocking group were shown to be Ser-Ser-Gln and an acetyl moiety, respectively. The N-terminal sequence of rat liver apoferritin is thus N-acetyl-Ser-Ser-Gln, which coincides with the N-terminal sequence of horse-spleen apoferritin, the only other apoferritin studied structurally at present.

In vitro stimulation of apoferritin synthesis by iron.

The apparent induction of apoferritin synthesis by iron has been examined in cell-free systems from rat and rabbit liver. Both systems allowed the complete synthesis de novo of apoferritin isolated by chromatographic or immunological means. Addition of iron at levels of 0.2--1 mM specifically stimulated incorporation of radioactive amino acids into apoferritin purified after classical heat extraction. The effect was also observed when iron was added at the end of the incubation period in the absence of continuing protein synthesis. Further, iron addition had no effect on the amount of newly synthesised apoferritin subunits as estimated by direct immunological precipitation from the reaction mixture. These results suggest that iron acts at some stage subsequent to translation in stimulating apoferritin biosynthesis.

Electron-microscopic studies on location of SH-groups in mitochondrial F1-ATPase using a ferritin label.

A new approach has been suggested for electron-microscopic study of the structure of mitochondrial F1-ATPase based on ferritin labeling. By means of sequential treatment with 2-iminothiolane and Nbs2 we obtained a modified ferritin (NbsSPrCNH-Ft) able to react with SH-groups of proteins and to form conjugates in which the protein and ferritin are bound by disulfide bonds. An electron-microscopic investigation of the negatively stained preparations of mitochondrial F1-ATPase, preincubated with modified ferritin, revealed such enzyme-ferritin conjugates. In case of modified ferritin, containing 360 mol SH-groups per mol protein, and F1-ATPase, pretreated with N-ethylmaleimide and then with dithiothreitol, conjugates were obtained in which ferritin molecules are bound to several (as many as four) of the six protein masses, comprising a bilayer molecule of the enzyme. Taking into consideration the biochemical data on the location of accessible SH-groups (only in alpha, gamma or epsilon subunits), it is inferred from the results obtained that one of the protein masses is a complex between beta subunit and at least one of the minor subunits located partially on the molecule's external side. This indicates the nonequivalence of different copies of the major subunits. Averaged images of the particles of the F1-F0 complex from bovine heart mitochondria and bacteria Micrococcus lysodeicticus were obtained. It was found that F0 component is bound to two adjacent protein masses of the F1-ATPase molecule. It is suggested that this binding may be due the nonequivalency of single-type major subunits.

Ultrastructural detection of fucosyl residues at the surface of axenically grown maize roots/sequential use of UeA lectin and fucosyl ferritin as the specific glycosylated marker.

This study reports the ultrastructural detection of fucosyl residues at the surface of axenically grown roots of Zea mays. The method used involved sequential binding of UeA lectin to the root and coupling of the bound UeA with fucosyl ferritin. Superficial dense ferritin labelling was found in the slime droplet ensheathing the root cap and in the external layer of the three-layered epidermal root surface. This pattern of binding of the fucose-specific lectin UeA suggests an overall distribution of the fucosyl residues on the root surface. Their localization within slime components of the root cap and the root epidermis is also assessed by the use of PATAg controls. Treatment of whole roots with alpha-L-fucosidase was ineffective in removing the fucosyl residues present at the root surface. The biological role of the fucosyl residues at the root-soil interface is discussed.

The amino acid sequence of human liver apoferritin.

The protein component of the iron storage molecule, ferritin, contains 24 subunits in form of a hollow shell known as apoferritin. The amino acid sequence has been determined for apoferritin subunits from human liver. The sequence comprises 174 amino acids giving an Mr of 19 900. It shows extensive homology with the primary structures of apoferritins from human and horse spleen and from rat liver. Sequence substitutions are discussed in relation to the known three-dimensional structure of horse spleen apoferritin. Evidence for a second minor sequence in human liver apoferritin is presented.

The location of antigenic sites on ferritin molecules.

Immunoreactivities of peptides purified after cleavage of human liver apoferritin are reported and discussed in relation to the known 3-dimensional and primary structures of homologous apoferritins. These studies point to 3 antigenic sites occupying continuous inter-helical regions of the polypeptide chains which lie on the surface of the apoferritin molecule. Other antigenic regions may encompass amino acids remote in the primary structure or belonging to different subunits.

The relationship between sialic acid content and peanut agglutinin binding on senescent and enzyme treated human erythrocytes.

Young, old and neuraminidase treated human red blood cells (RBC) were investigated with peanut agglutinin (PNA), a lectin with a specificity similar to that of serum T-agglutinin. The effect of serum agglutinins on this interaction was also investigated. The density and distribution of PNA receptors were evaluated by agglutination with PNA and binding of ferritin-conjugated PNA (PNA-F), or PNA labeled with radioactive iodine [( 131I] PNA). The results were correlated with the distribution of membrane bound sialic acids, as evaluated by chemical analysis and rate of agglutination with poly-L-lysine (PLL). Untreated RBC of all ages did not agglutinate with PNA and failed to bind PNA-F and [131I] PNA. Treatment of young RBC with neuraminidase, which resulted in reduction of membrane-bound sialic acids to an extent similar to that of physiologically aged RBC, resulted in the concomitant exposure of PNA binding sites and in the agglutination of these cells by autologous serum. Pretreatment of the neuraminidase treated RBC with autologous serum resulted in partial inhibition of the binding capacity of PNA on the RBC. The results indicate that the normal age-related loss of sialic acids in circulating RBC is not identical with enzymatic removal of sialic acids by neuraminidase. The observations suggest that different mechanisms are functional in the recognition and sequestration of old RBC and of RBC treated with neuraminidase.

Distribution of sialoglycoconjugates on the luminal surface of the endothelial cell in the fenestrated capillaries of the pancreas.

Sialic acid-bearing molecules on the luminal surface of the vascular endothelium in mouse and rat pancreatic capillaries were detected electron microscopically by using a procedure with ferritin hydrazide (FH), after preferential oxidation of sialyl residues with sodium periodate. The distribution of FH on the endothelial surface demonstrated the existence of microdomains with various densities of sialoglycoconjugates oxidizable by sodium periodate and accessible to the tracer. On the plasmalemma proper, FH binding sites were heterogeneously distributed. Their concentration on various microdomains decreased as follows: plasmalemma proper greater than coated pits greater than stomal diaphragms of plasmalemmal vesicles and transendothelial channels, and fenestral diaphragms. The membrane of plasmalemmal vesicles and transendothelial channels was not labeled by FH. Nonspecific binding of FH to the nonoxidized endothelial surface or that oxidized after neuraminidase treatment was relatively low.

Synthesis of apoferritin in mouse peritoneal macrophages. Characterization of 20 S particles.

Apoferritin particles were found in mouse peritoneal macrophages cultured in vitro. They were found as 20S particles in the "ribosomal fraction" of macrophages labeled with L-[14C]glutamic acid. Possibilities that they were breakdown products of ribosomes or of other well-known contaminants of the ribosomal fraction were excluded because they did not incorporate [5-3H]uridine. They were resistant to RNase and were relatively resistant to detergent. The antibody against horse spleen apoferritin precipitated about 70% of the particles in the 20S region, judging by measurement of radioactivity. On in vitro incubation with Fe2+ and suitable oxidizing agents the sedimentation coefficient of 80% of the 20S particles changed to about 60S, which corresponds to that of ferritin. SDS-polyacrylamide gel electrophoresis revealed the presence of subunit structures with the same molecular size as that of mouse liver apoferritin. Under the electron microscope, the particles appeared spherical with a relatively uniform diameter of about 130 A.

Glycosylated serum ferritin in patients with hematological malignancies before and after bone marrow transplantation.

Glycosylated and total serum ferritin levels were monitored in patients with acute leukemia and lymphoma undergoing bone marrow transplantation (BMT). Serum ferritin was high in relapsing patients and normal in most patients in complete remission (CR). In patients with an uncomplicated course, levels of ferritin increased during the first month after BMT with subsequent decrease. Three patients with lymphoma and five with acute leukemia had high serum ferritin levels despite achieving apparent complete hematological remission which was of short duration. The results were compared with groups of lymphoma patients at presentation and during remission and with healthy normal controls. In all the lymphoma patients and in 3 of the 5 leukemia patients the percent of ferritin glycosylation was normal at CR. It was low at the time of diagnosis in all patients. Thus, the percent glycosylation proved a more reliable marker for clinical remission than total serum ferritin. During follow up after BMT in uncomplicated cases, the percent of glycosylated ferritin returned to normal levels earlier than the total serum ferritin. These findings indicate that the evaluation of the amount of glycosylated ferritin may provide useful information in hematological patients in whom there is a discrepancy between high serum ferritin levels and the clinical condition.

Spectroscopic studies on the binding of iron, terbium, and zinc by apoferritin.

Ultraviolet difference spectroscopy has been used to study Fe (III)-apoferritin complexes formed after addition of Fe (II) to apoferritin in air. At constant iron, the recorded spectra varied with time after Fe (II) addition and with the number of iron atoms/molecule (protein concentration). The results indicate that after production of an initial complex, rearrangement or migration of Fe (III) atoms occurs, with polynuclear species forming as end-product, probably by hydrolytic polymerization. The presence of Tb3+ or Zn2+ ions affected the Fe (III) spectra and their development in different ways. The combined data suggest that more than one site, or processes, are involved in ferritin iron-core formation and that some of the metal sites are clustered.

Immunoradiometric and electroimmuno assay of increased ferritin and apoferritin levels in serum.

Ferritin in serum from patients with increased serum ferritin levels has been studied both quantitatively and qualitatively. All techniques utilized in these studies are suitable to be used as routine screening tests for large numbers of patients. Electroimmuno assay (EIA) has been compared with the solid phase immunoradiometric (IRMA) assay as a technique to determine serum ferritin concentration (r = 0.99) and is suggested as a useful alternative when determining ferritin concentrations above 500 microgram/l. Iron stained EIA gels have been used to indicate the iron content of the ferritin molecule in sera. This simple screening test has demonstrated that apoferritin is found more often than iron-rich ferritin in the serum of patients with elevated serum ferritin levels. Immunoelectrophoresis precipitin bands suggest the heterogeneity of ferritin in serum from different patients.

[The serum and intraerythrocyte concentration of ferritin in the anemia of rheumatoid arthritis]. / Concentración sérica e intraeritrocitaria de ferritina en la anemia de la artritis reumatoide.

Serum concentration of ferritin (Ft) and its glycosylated fraction (Ft-Gl) and intraerythrocytic ferritin (Ft-e) concentration were measured in 26 patients with anemia and active rheumatoid arthritis. Patients were divided into 2 groups according to the presence of anemia of chronic diseases (n = 13) or associated ferropenia. Unlike the first group, patients with associated ferropenia had lower concentration of the above parameters than 31 control subjects. The logarithmic value of FT (log FT) directly correlated with globular sedimentation velocity. Ft-Gl and log Ft-e correlated with transferrin saturation (r = 0.603, p less than 0.01 and r = 0.444, p less than 0.05). Log Ft-e also correlated with Ft (r = 0.504, p less than 0.01). The probability of ferropenia when Ft was 60 micrograms/l or lower was 0.91, and when Ft-e was 1.5 ag/cel or lower was 0.66. It is concluded that the ferropenic status in active rheumatoid anemia decreases the iron dependent synthesis of ferritin (Ft-Gl) more than that mediated by the acute phase response. The intraerythrocytic content is low due to the scanty iron supply to the erythroblast. Ft is more efficacious than Ft-e in the diagnosis of ferropenia.

[Age-related changes in concentrations of ferritin, glyeosylated ferritin, and non-glycosylated ferritin].

We studied age-related changes in the concentrations in serum of ferritin, glycosylated ferritin, and non-glycosylated ferritin. The concentrations were determined in 95 healthy subjects: 39 men and 56 women, aged from 22 to 94 years. In the men, age correlated significantly with serum ferritin (r = 0.332, p < 0.05) and non-glycosylated serum ferritin (r = 0.628, p < 0.001) but not with glycosylated serum ferritin. In the women, age correlated significantly with serum ferritin (r = 0.456, p < 0.001), non-glycosylated serum ferritin (r = 0.439, p < 0.001), and glycosylated serum serum ferritin (r = 0.415, p < 0.01). The ratio of glycosylated serum ferritin to serum ferritin correlated negatively with age both in men and in women (men: r = -0.661, p < 0.001; women: r = -0.411, p < 0.01). Serum non-glycosylated ferritin levels were higher in older men. Both serum glycosylated ferritin and non-glycosylated ferritin levels were higher in older women, but this phenomenon was more pronounced with respect to the non-glycosylated form. These results suggest that hyperferritinemia in the elderly is mainly caused by an increase in the concentration of non-glycosylated ferritin, both in men and in women.

[High levels of serum ferritin in elderly patients with non-insulin-dependent diabetes mellitus].

We studied concentrations of serum ferritin, glycosylated ferritin, and non-glycosylated ferritin in elderly patients with diabetes. The subjects were 111 people who were at least 60 years old: 54 healthy controls, 14 diabetic patients without retinopathy, and 43 diabetic patients with retinopathy. The mean levels of ferritin, glycosylated ferritin, and non-glycosylated ferritin in serum were significantly higher in the patients with retinopathy than in healthy controls. The mean percent glycosylated ferritin did not differ between patients with retinopathy and healthy controls. The mean levels of serum ferritin, glycosylated ferritin, and non-glycosylated ferritin, and the percent glycosylated ferritin did not differ significantly between patients without retinopathy and health controls. None of these values differed between subjects with macroangiopathy and those without macroangiopathy, in both groups of patients. In patients with diabetes, none of the values measured was significantly related to fasting plasma glucose, HbA1c, or the duration of diabetes. These results suggest that diabetic microangiopathy is associated with abnormally high levels of ferritin in serum.