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E C Theil

Publications and source records attributed to E C Theil.

At least 73 records · Page 4Linked to original sources
PubMed1986

Multiple red cell ferritin mRNAs, which code for an abundant protein in the embryonic cell type, analyzed by cDNA sequence and by primer extension of the 5'-untranslated regions.

Ferritin maintains iron in a bioavailable, nontoxic form for vertebrates and invertebrates, higher plants, fungi, and bacteria; the protein is formed from two classes of subunits (H and L) in ratios which vary in different cell types. Ferritin may be an abundant, differentiation-specific protein or a "housekeeping" protein. The red cells of embryos are specialized for iron storage and have abundant ferritin; iron regulates the synthesis of ferritin in such cells translationally by recruitment of stored, ferritin mRNA and by translational competition. To characterize mRNA regulated in such a manner, we prepared cDNA from reticulocytes of bullfrog tadpoles, a readily available source of embryonic red cells; moreover, no protein sequence information was available for nonmammalian ferritin. An almost full-length (817 base pairs) cDNA (pJD5F12) was isolated and sequenced, the 5' end was analyzed by primer extension, and the cloned DNA was used as a hybridization probe. We have shown that ferritin mRNA is stored in the cytoplasm and that the 5' end of the mRNA is heterogeneous. The 5'-untranslated region of ferritin mRNA consisted of 143 nucleotides in the major (65%) species and 146 or 152 in the minor species (approximately 17% each). (Heterogeneity is characteristic of some other abundant mRNAs, e.g. globin, which is also translationally regulated.) Since excess iron had no detectable effect on the heterogeneity of the 5' end of ferritin mRNA, the feature is more likely associated with mRNA abundance and/or cell specialization than translational control. In the bullfrog, as in humans and rats, ferritin is encoded by multiple genomic sequences (four to eight) which specify proteins of considerable homology. For example, 75 of the 81 amino acids present in all mammalian ferritins sequenced are also present in the frog; the overall homology between frogs and humans or rats is 59-66%. Ferritin H and L subunits in humans are distinct (overall homology 56%) and appear to have diverged from a common precursor relatively recently. In contrast, ferritin H and L subunits have high homology in tadpole red cells, determined by hybrid select translation, which suggests that bullfrog red cell ferritin may be close to the primordial sequence.

Amino Acid Sequence↗
PubMed1986

Structural variations in soluble iron complexes of models for ferritin: an x-ray absorption and Mössbauer spectroscopy comparison of horse spleen ferritin to Blutal (iron-chondroitin sulfate) and Imferon (iron-dextran).

Variations in the turnover of storage iron have been attributed to differences in apoferritin and in the cytoplasm but rarely to differences in the structure of the iron core (except size). To explore the idea that the iron environment in soluble iron complexes could vary, we compared horse spleen ferritin to pharmaceutically important model complexes of hydrous ferric oxide formed from FeCl3 and dextran (Imferon) or chondroitin sulfate (Blutal), using x-ray absorption (EXAFS) and Mössbauer spectroscopy. The results show that the iron in the chondroitin sulfate complex was more ordered than in either horse spleen ferritin or the dextran complex (EXAFS), with two magnetic environments (Mössbauer), one (80%-85%) like Fe2O3 X nH2O (ferritinlike) and one (15%-20%) like Fe2O3 (hematite); since sulfate promotes the formation of inorganic hematite, the sulfate in the chondroitin sulfate most likely nucleated Fe2O3 and hydroxyl/carboxyls, which are ligands common to chondroitin sulfate, ferritin and dextran most likely nucleated Fe2O3 X nH2O. Differences in the structure of the iron complexed with chondroitin sulfate or dextran coincide with altered rates of iron release in vivo and in vitro and provide the first example relating function to local iron structure. Differences might also occur among ferritins in vivo, depending on the apoferritin (variations in anion-binding sites) or the cytoplasm (anion concentration).

Animals↗
PubMed1985

Fe(III).ATP complexes. Models for ferritin and other polynuclear iron complexes with phosphate.

Polynuclear iron complexes of Fe(III) and phosphate occur in seawater and soils and in cells where the iron core of ferritin, the iron storage protein, contains up to 4500 Fe atoms in a complex with an average composition of (FeO.OH)8FeO.OPO3H2. Although phosphate influences the size of the ferritin core and thus the availability of stored iron, little is known about the nature of the Fe(III)-phosphate interaction. In the present study, Fe-phosphate interactions were analyzed in stable complexes of Fe(III).ATP which, in the polynuclear iron form, had phosphate at interior sites. Such Fe(III).ATP complexes are important not only as models but also because they may play a role in intracellular iron transport and in iron toxicity; the complexes were studied by extended x-ray absorption fine structure, EPR, NMR spectroscopy, and measurement of proton release. Mononuclear iron complexes exhibiting a g' = 4.3 EPR signal were formed at Fe:ATP ratios less than or equal to 1:3, and polynuclear iron complexes (Fe greater than or equal to 250, EPR silent at g' = 4.3) were formed at an Fe:ATP ratio of 4:1. No NMR signals due to ATP were observed when Fe was in excess (Fe:ATP = 4:1). Extended x-ray absorption fine structure analysis of the polynuclear Fe(III).ATP complex was able to distinguish an Fe-P distance at 3.27 A in addition to the octahedral O at 1.95 A and 4-5 Fe atoms at 3.36 A. The Fe-O and Fe-Fe distances are the same as in ferritin, and the Fe-P distance is analogous to that in another metal-ATP complex. An observable Fe-P environment in such a large polynuclear iron cluster as the Fe(III).ATP (4:1) complex indicates that the phosphate is distributed throughout rather than merely on the surface, in contrast to earlier models of chelate-stabilized iron clusters. Complexes of Fe(III) and ATP similar to those described here may form in vivo either as normal components of intracellular iron metabolism or during iron excess where the consequent alteration of free nucleotide triphosphate pools could contribute to the observed toxicity of iron.

Adenosine Triphosphate↗
PubMed1984

Developmental changes in plasma transferrin concentrations related to red cell ferritin.

Transferrin, the plasma iron transport protein, has two iron-binding sites but is usually only partly saturated. (Essentially all plasma iron is bound to transferrin.) Thus, changes in transferrin saturation reflect differences in the concentrations of plasma iron and/or transferrin. Developmental changes in red cell ferritin content coincide with a 2.5 times increase in plasma transferrin and a proportional decrease in saturation, in the bullfrog model system. The possible relationship of the degree of transferrin saturation and structure on the distribution of iron between heme and ferritin was examined in suspensions of reticulocytes, which synthesize both ferritin and heme. The extra transferrin in adult plasma was indistinguishable from transferrin in tadpole plasma in terms of the ability to donate iron to red cell heme and ferritin in vitro and in terms of surface charge (pI 6.55, 6.34), molecular weight (73,000), carbohydrate content (2%), amino acid composition, and immunological reactivity. Only the saturation in vivo appeared to differ. When the saturation of transferrin was manipulated in vitro, an effect on the relative distribution of iron between heme and ferritin was observed. The heme-synthesizing system consumed a disproportionately large amount of the delivered iron until it was saturated, a point which coincided with transferrin saturation; as the degree of transferrin saturation decreases, iron delivered to red cell iron stores (ferritin) decreases disproportionately. Thus, the developmental increase in plasma transferrin and consequent decrease in saturation minimize the amount of iron available for storage in red cells. The effect is further enhanced by the decreased ability of adult erythrocytes to incorporate iron from transferrin, a property which may be related to quantitative changes observed in iodination of a Mr = 168,000 membrane protein.

Age Factors↗
PubMed1984

Cell-specific properties of red cell and liver ferritin from bullfrog tadpoles probed by phosphorylation in vitro.

Cell-specific differences occur in the primary structure of ferritin. For example, red cell and liver ferritin from bullfrog tadpoles differ by 1.5 times in serine content. To determine if cell-specific differences in ferritin primary structure are expressed in the tetraeicosomer, which thus might distinguish the proteins in a functional state, phosphorylation in vitro was employed as a probe using [gamma-32P]ATP and the catalytic subunit from the cAMP-dependent protein kinase of bovine skeletal muscle. Subunits of both proteins in the tetraeicosomers were phosphorylated. Based on tryptic peptide maps, five regions common to both red cell and liver apoferritin were phosphorylated, as confirmed for two peptides by amino acid analyses. [32P]Apoferritin from red cells yielded an additional four 32P-fragments by mapping, at least three of which were unique by amino acid analysis and, in one case, might represent a 32P-Fe complex bound by a fragment of the iron-binding site. One peptide appeared to be unique to liver apoferritin. High concentrations of ATP yielded one additional peptide common to liver and red cell and one red cell-specific peptide in the tryptic peptide maps. The maximum moles of 32P/molecule were 13 +/- 4 and 6 +/- 2, respectively, for red cell and liver apoferritin, which corresponded within experimental error to the number of 32P-tryptic peptides. The level of phosphorylation was, on the average, not more than one site/subunit. Furthermore, above certain levels of phosphorylation, some subunits in the assemblage of 24 appeared to be unavailable as substrates, possibly because of charge repulsion or conformational changes. The possibility that post-translational modifications of ferritin which amplify cell-specific structural features occur in vivo with cytoplasmic components, e.g. protein kinases, is considered in terms of the physiological availability of iron from different iron storage cells and developmental changes in iron storage.

Animals↗
PubMed1983

A distinct environment for iron (III) in the complex with horse spleen apoferritin observed by x-ray absorption spectroscopy.

Cell-specific variations in apoferritin structure correlate with variations in iron metabolism that suggest functional specificity of the protein shell. Using EPR spectroscopy, we previously showed that vanadyl binds to specific sites on apoferritin, and that VO2+ binding is reduced by Fe(II) and Fe(III) (the natural substrates) and by metals known to influence iron storage (Chasteen, N. D., and Theil, E. C. (1982) J. Biol. Chem. 257, 7672-7677). Such observations suggest that the metal-binding site is important to apoferritin function and may define a location where the influence of cell-specific structural features are exerted. To investigate the iron-protein complex further, we have used x-ray absorption spectroscopy and have characterized, for the first time to our knowledge, Fe(III) apparently attached to the protein, after analyzing the x-ray absorption spectrum of an Fe(III)-apoferritin complex (10 Fe/molecule) compared to that of ferritin (polynuclear Fe(III)OOH, about 2000/molecule). The environment of iron in the Fe(III)-protein complex was similar to that in an Fe(III)-oxalate (2:3) hexahydrate complex, both in near edge structure and extended x-ray absorption structure, confirming earlier predictions of carboxylates as protein ligands. The extended x-ray absorption fine structure data for both compounds was fit best by a model in which a second shell of low Z atoms (carbon) was close (0.53-0.55 A) to the first shell of coordinated oxygen. However, small differences between Fe(III)-apoferritin and Fe(III)-oxalate in the Fe-O environment suggest a distorted geometry in the Fe(III)-protein complex and/or the presence of a mixture of atoms, such as nitrogen and oxygen, coordinated to iron. Extension of this approach to other apoferritins and metals will be likely to clarify the role of cell-specific features of the apoprotein in the formation of the iron core.

Animals↗
PubMed1983

Subunit dimers in sheep spleen apoferritin. The effect on iron storage.

Ferritin with high and low iron content, 2000 and 790 iron atoms/molecule, was isolated from the spleens of copper-poisoned and control lambs, respectively. Differences in the iron content in vivo were reflected in the properties of the apoferritin protein shells, since the apoprotein from the low iron ferritin took up iron relatively more slowly (0.52 +/- 0.09) and released it more rapidly (1.68 +/- 0.06) in vitro. Although the two types of apoferritin were indistinguishable in terms of surface charge (pI range 4.98-5.43) and in consisting of both heavy and light subunits, the subunit interactions differed markedly; 40-50% of the subunits of low iron ferritin were in dimers stable to reduction and carboxylmethylation, 4% mercaptoethanol, 8% sodium dodecyl sulfate, and 100 degrees C for 30 min, 70% formic acid, and 30% methanol. Subunit dimers were also observed in liver ferritin from mouse and neonatal pig and were enriched in a low iron fraction of horse spleen ferritin. Based on cyanogen bromide fragmentation and NH2-terminal analysis, the natural and chemically cross-linked subunit dimers had two peptides in common; natural subunit dimers also appeared to have a second region cross-linked, suggesting the possibility of both intra- and intersubunit links in the natural dimers. In sheep spleen ferritin, both heavy and light subunits appeared to participate in subunit dimerization. Natural subunit dimers were enriched in low iron ferritin fractions of all ferritin preparations tested (linear correlation = 0.94) and can explain, at least in part, the previously observed effects of iron core size on the apoferritin shell. Whether the subunit cross-links represent part of the subunit assembly process subsequently cleaved by iron (or copper) or whether the cross-links form after iron core formation in vivo has yet to determined. In either case, it is clear that such post-translational variations can affect iron uptake and release and emphasize the importance of the protein shell in determining the iron storage properties of ferritin.

Amino Acids↗
PubMed1983

Regulation of ferritin mRNA: a possible gene-sparing phenomenon. Induction of ferritin synthesis by iron in liver as well as red cells combines high translational efficiency with increased utilization of preformed ferritin mRNA.

Control of ferritin synthesis by iron at the level of transcription is potentially hazardous to DNA because of the iron-catalyzed degradation of DNA. The induction of ferritin synthesis in reticulocytes of embryos (bullfrog tadpoles) occurs by two types of translational control i.e. increased availability of stored ferritin mRNA, in response to iron, coupled with a high translational efficiency. Since erythroid cell nuclei have large amounts of heterochromatin and may be relatively inactive genetically, the translational control of ferritin by iron observed in red cells was studied in other tissue by isolating poly (A+) RNA from tadpole liver and analyzing protein synthesis in vitro. Liver ferritin mRNA directed the synthesis of 7.0% of the protein in a wheat germ system, compared to 1.2% in vivo, suggesting that tadpole liver contained a large amount of stored ferritin mRNA. At levels of poly (A+) RNA which were saturating for total protein synthesis, ferritin synthesis was still linearly dependent upon RNA concentration, indicating a high efficiency of translation of ferritin mRNA. The results are analogous to those previously observed in red cells and confirm the storage of ferritin mRNA deduced from studies of the polysomal and nonpolysomal distribution of the mRNA in rat liver. The results indicate that the increased availability for translation of stored ferritin mRNA, in response to iron, and the high translational efficiency of ferritin mRNA are a general characteristic of ferritin synthesis rather than a specific feature of red cell maturation. This novel form of regulation of ferritin gene expression can be attributed to a need to protect DNA from degradation by iron and oxygen. The normal barrier between DNA and iron is apparently breached by the iron-oxygen complex of the drug bleomycin, an antitumor agent thought to act in vivo by iron-catalyzed cleavage of DNA.

Animals↗
PubMed1982

Translational control of ferritin synthesis by iron in embryonic reticulocytes of the bullfrog.

The regulation of ferritin synthesis by iron was examined in the reticulocytes of bullfrog tadpoles where the induction was 40- to 50-fold, increasing from 0.17 +/- 0.05% of total protein synthesis ([3H]leucine incorporation in cell suspension) to 7.4 +/- 1.6% following intraperitoneal injection of ferric ammonium citrate. No significant difference was observed between the levels of ferritin mRNA in control or iron-induced cells, determined by translation of isolated mRNA in a wheat germ system, demonstrating that ferritin induction by iron occurs by a post-transcriptional mechanism. Total protein synthesis in the wheat germ system was half-saturating at 10 micrograms of mRNA/ml whereas ferritin synthesis increased linearly up to 40 micrograms of mRNA/ml, demonstrating that the ferritin mRNA is translated with high efficiency relative to the total proteins synthesized. Studies with the cap analogue 7-methylguanosine-5'-monophosphate, suggest that cap binding is not directly involved in the high translational efficiency of the ferritin mRNA in the wheat germ system. The results indicate that iron-modulated changes in the availability of ferritin mRNA for translation, coupled with the high translational efficiency of the ferritin message, can account for the induction of ferritin synthesis by iron in embryonic erythroid cells.

Animals↗
PubMed1979

Similarity of the structure of ferritin and iron . dextran (imferon) determined by extended X-ray absorption fine structure analysis.

Ferritin, a natural complex of iron oxide encased in protein, and iron . dextran, a synthetic complex of iron oxide coated with dextran, have the similar properties of maintaining high concentrations of iron in solution at physiological pH and releasing iron relatively slowly in vivo. Extended x-ray absorption fine structure (EX-AFS) analysis was performed on each complex and compared to see if the structures of the iron cores were similar. The results obtained from the extended x-ray absorption fine structure technique show that the near-neighbor environment around the average iron atom in ferritin and iron . dextran is identical, within experimental uncertainty, for the first three shells. The similarity of the iron cores in both complexes may explain the similarity of iron release in vivo. Ferritin has a protein coat which is composed of 24 subunits arranged in a hollow sphere with six channels through which the iron may move during deposition and release. However, little is known about the requirements of the protein structure in ferritin for the maintenance of high concentrations of iron in a soluble, nontoxic form or about the role of the protein in the release of iron from ferritin. The results suggest that iron . dextran will be a useful model compound in studies of the relation of the iron core and protein in ferritin to function.

Dextrans↗
PubMed1978

The induction of ferritin synthesis in circulating larval red blood cells.

The circulating red blood cells formed in bullfrog larvae, chicken embryos, and mouse embryos contain large amounts of ferritin and storage iron in excess of the need for hemoglobin. In contrast, the circulating red cells of adult animals contain little ferritin. Ferritin synthesis and iron storage are coordinated with differentiation and hemoglobin synthesis in the red cells of adults. In order to test the hypothesis that ferritin synthesis could be controlled independently of hemoglobin synthesis and differentiation in the red cells formed early in life, bullfrog larvae were injected with iron to determine if ferritin synthesis was increased in the circulating red cells. Within 17 h after the injection of iron, the synthesis of ferritin, assayed as the incorporation of [14C]leucine by cell suspensions prepared from circulating red cells, was increased from 2.9 to 10.2% of the total protein, and the specific activity of the ferritin synthesized increased from 1100 to 3000 cpm/A280. There was no change in the hematocrit of the animals nor in the specific activity of hemoglobin synthesized by suspensions of red cells (average, 720 cpm/A280). The results suggest that in mature, larval red cells, ferritin synthesis can be controlled by changes in the extracellular environment. The results also indicate that ferritin synthesis can be controlled independently of hemoglobin synthesis with which it is coordinated during erythroid differentiation in adult animals.

Animals↗