Tritrichomonas foetus: ultrastructure and cytochemistry of endocytosis

Tritrichomonas foetus ingests horseradish peroxidase, native ferritin, cationized ferritin, and 0.08 micron latex beads by a process which involves the formation of pinocytic vesicles. These vesicles fuse with each other and with lysosomes forming large vacuoles. Biochemical determinations on the ingestion of horseradish peroxidase and morphometric analysis on the ingestion of cationized ferritin covered latex beads indicated that T. foetus has high endocytic activity. The process of ingestion of the various tracers used was analyzed by transmission electron microscopy of thin sections and freeze fracture replicas.     

Envelope of Mouse Mammary Tumor Virus Studied by Freeze-Etching and Freeze-Fracture Techniques

As part of a study of the cell surface changes associated with the production of murine mammary tumor virus, the structure of the envelope of this virus has been examined by using freeze-fracture techniques. Both fracture and deep-etch surfaces were examined. The fracture faces contain 10-nm spheres comparable to those observed on fractured plasma membranes, although fewer in number. Surfaces exposed by etching possess a highly regular hexagonal array of pits 25 nm apart. By examining freeze-fracture and freeze-etch preparations of virus with ferritin covalently bound to its surface, it has been determined that the surface exposed by etching is the outer surface of the virus. The pitted exterior surface of the mammary tumor virus appears to be a unique surface structure.     

Oriented adsorption of purple membrane to cationic surfaces

We have investigated the orientation of isolated fragments of Halobacterium halobium purple membrane (PM) adsorbed to poly-L-lysine- treated glass (PL-glass), by quanitative electron microscopy. Three lines of evidence support the conclusion that the cytoplasmic side of the membrane is preferentially absorbed. First, monolayer freeze- fracture reveals nonrandom orientation; more fracture faces (89%) are particulate than smooth. Second, the amount of each membrane surface present can be assayed using polycationic ferritin; 90% of all adsorbed membrane fragments are labeled. Third, it is possible to distinguish two surfaces, "cracked" (the extracellular surface) and "pitted" (the cytoplasmic surface) , in slowly air-dried, platinum-carbon-shadowed membranes. When applied under standard conditions, more than 80% appear cracked. Selection for the cytoplasmic by the cationic substrate suggests that the isolated PM, buffered at pH 7.4 and in the light, has a higher negative charge on its cytoplasmic surface than on its extracellular surface. Nevertheless, cationic ferritin (CF) preferentially adsorbs to the extracellular surface. Orientation provides a striking example of biomembrane surface asymmetry as well as the means to examine the chemical reactivity and physical properties of surfaces of a purified, nonvesicular membrane fragment.     

A long term survey of plasma and tissue ferritin in mice fed on iron deficient diets

1. Changes in plasma and tissue ferritin concentrations were surveyed in mice fed on iron deficient diets (Fe, 2.4 micrograms/g) from the age of 8 to 30 weeks by an enzyme-immunoassay for mouse ferritin. Values were compared with those in mice on control diets (Fe, 110 micrograms/g) and mice with blood loss. 2. In mice on iron deficient diets, while plasma ferritin remained at normal level, ferritin in the spleen and kidney decreased, but that in the liver increased as much as twice of that in the control. 3. Mice with blood loss revealed a drastic reduction of ferritin in every tissue and plasma examined.     

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.     

Characterization of the binding of ferritin to the rat liver ferritin receptor

The binding characteristics and specificity of the rat hepatic ferritin receptor were investigated using ferritins prepared from rat liver, heart, spleen, kidney and serum, human liver and serum, guinea pig liver and horse spleen as well as ferritins enriched with respect to either H- or L-type subunit composition, prepared by chromatofocusing of rat liver ferritin on Mono-P or by reverse-phase chromatography of ferritin subunits on ProRPC 5/10. No significant difference was apparent in the binding of any of the tissue ferritins, or of ferritins of predominantly acidic or basic subunit composition. However, serum ferritin bound with a lower affinity. The effect of carbohydrate on the ferritin-receptor binding was examined by glycosidase treatment of tissue and serum ferritins. Tissue ferritin binding was unaffected, while serum ferritin binding affinity was increased to that of the tissue ferritins. Inhibition of ferritin binding by lactoferrin was not due to common carbohydrate moieties as previously suggested but was due to direct binding of lactoferrin to ferritin. Therefore, carbohydrate residues do not appear to facilitate receptor-ferritin binding, and sialic acid residues present on serum ferritin may in fact interfere with binding. The results indicate that the hepatic ferritin receptor acts preferentially to remove tissue ferritins from the circulation. The lower binding affinity of serum ferritin for the ferritin receptor explains its slower in vivo clearance relative to tissue ferritins.     

Characterization of the binding of ferritin to the rat liver ferritin receptor

The binding characteristics and specificity of the rat hepatic ferritin receptor were investigated using ferritins prepared from rat liver, heart, spleen, kidney and serum, human liver and serum, guinea pig liver and horse spleen as well as ferritins enriched with respect to either H- or L-type subunit composition, prepared by chromatofocusing of rat liver ferritin on Mono-P or by reverse-phase chromatography of ferritin subunits on ProRPC 5/10. No significant difference was apparent in the binding of any of the tissue ferritins, or of ferritins of predominantly acidic or basic subunit composition. However, serum ferritin bound with a lower affinity. The effect of carbohydrate on the ferritin-receptor binding was examined by glycosidase treatment of tissue and serum ferritins. Tissue ferritin binding was unaffected, while serum ferritin binding affinity was increased to that of the tissue ferritins. Inhibition of ferritin binding by lactoferrin was not due to common carbohydrate moieties as previously suggested but was due to direct binding of lactoferrin to ferritin. Therefore, carbohydrate residues do not appear to facilitate receptor-ferritin binding, and sialic acid residues present on serum ferritin may in fact interfere with binding. The results indicate that the hepatic ferritin receptor acts preferentially to remove tissue ferritins from the circulation. The lower binding affinity of serum ferritin for the ferritin receptor explains its slower in vivo clearance relative to tissue ferritins.     

Local conformational changes in ferritins with variable iron content

Conformational changes were induced in human spleen ferritin by partial or complete removal of iron, and the immunoreactivity of the ferritin samples with variable iron content was analyzed. We established that a decrease in iron content resulted in bimodal changes in immunoreactivity of the epitopes recognized by the monoclonal antibodies G10 and F11. Immunoreactivity demonstrated a 3-6-fold decrease on lowering iron content from 800 to 40 atoms per protein molecule, followed by a sharp (4-14-fold) increase that was observed when low-iron ferritin was converted to iron-free apoferritin. These bimodal changes suggest the presence of more than two conformational states of ferritin with local alterations of the epitopes recognized by the monoclonal antibodies. The global conformation of ferritin, however, remained essentially unaltered, as demonstrated by ferritin interaction with polyclonal antibodies. Together, the results indicate that local conformational changes in the ferritin protein shell occur on progressive iron removal that results in low-iron and iron-free forms of ferritin. These changes are most clearly seen in apoferritin when compared to low-iron ferritin.     

Differential effects of iron and inflammation on ferritin synthesis on free and membrane-bound polyribosomes of rat liver.

We have examined the distribution of ferritin mRNA to free and endoplasmic reticulum (ER)-bound liver polyribosomes during inflammation and iron treatment of rats. Postnuclear tissue supernatants were fractionated on a discontinuous sucrose gradient developed to separate free and bound polyribosomes. Total RNA recovered averaged 3.2 mg/g tissue, 40% of which was with ER and 30% with the free polyribosomes, about 25% being with the postribosomal/RNP fraction. Slot-blot hybridization of equal portions of RNA revealed that 12 h after injection of turpentine to induce inflammation, ferritin mRNA was concentrated on the ER-bound polyribosomes, while it was concentrated on the free polyribosomes 2 h after injection of ferric ammonium citrate. Differences were highly significant, based on multiple determinations and densitometry. Profiles of ferritin mRNA distribution on linear sucrose gradients corroborated the differential findings. Concentrations of total ferritin mRNA per gram liver doubled with iron treatment but were not significantly different 12 h after turpentine treatment. At the same time point after turpentine, ferritin protein synthesis was increased twofold, as measured by the 1 h incorporation of [14C]leucine. We conclude that a significant portion of ferritin mRNA always associates with the ER-bound polyribosomes, and that inflammation and iron differentially alter the polysomal distribution of ferritin mRNA, suggesting that two different kinds of mRNA may be involved.     

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.     

Hydrogen ion interactions of horse spleen ferritin and apoferritin.

The interactions of horse spleen ferritin and its derivative apoferritin with H+ ions were studied by potentiometric and spectrophotometric titration; to aid in data analysis, heats of ionization over a limited pH range and amide content were also determined. Per apoferritin subunit, all tyrosine and cysteine side chains, two of the nine lysine side chains and at least three of the six histidine side chains were found not to titrate; a preliminary but self-consistent analysis of the titration data is proposed. The titration curve of ferritin was identical with that of apoferritin in the pH range 5.5 to 3. In addition, under the conditions used, the reactivities of ferritin histidines to bromoacetate and of ferritin lysines to formaldehyde were identical with those in apoferritin. Above pH 8, a time-dependent titration of the ferritin core occurs which prevents comparison of the titration curves of the two proteins in this region. However, in the pH regions 5.5 to 7.5, two extra groups per subunit titrate reversibly in ferritin relative to apoferritin. Moreover, although the isoionic points of ferritin and apoferritin are identical in water, the isoionic point of ferritin is 0.5 pH unit lower than that of apoferritin in 0.16 to 1 M KCl. The different effects of KCl and NaCl on the two proteins indicate the presence of cation binding sites in ferritin that are absent in apoferritin and possibly also the presence of anion binding sites in apoferritin that are occupied in ferritin by anions of the core. The difference between the isoionic points of the two proteins in KCl has been interpreted to indicate the presence of approximately 2 phosphate residues per ferritin subunit which serve as cation binding sites and which are negatively charged at the isoionic point in KCl. These phosphates may also represent the additional residues that titrate in ferritin between pH 5.5 and 7.5, or may interact with positively charged residues on the inner surface of the ferritin shell, or both.     

Purification and characterization of a small dermatan sulphate proteoglycan implicated in the dilatation of the rat uterine cervix.

Ferritin was purified from chicken liver by two different methods: gel filtration on controlled-pore glass beads, and immunoaffinity chromatography employing a chicken ferritin-specific monoclonal antibody that did not cross-react with horse spleen ferritin. This antibody recognizes intact ferritin and an oligomeric 240 kDa form of the molecule after protein transfer to nitrocellulose, but not the 22 kDa chicken ferritin subunit. Chicken liver ferritin purified by these methods exhibited reduced migration on non-denaturing polyacrylamide gels compared with horse spleen ferritin. These results were consistent with the difference in calculated isoelectric points of chicken and horse ferritin subunits. By two-dimensional gel electrophoresis, chicken ferritin 22 kDa subunits exhibited isoelectric points from 6.1 to 6.6 whereas horse spleen ferritin subunits exhibited isoelectric points of 5.8-6.3. The 240 kDa form of the chicken ferritin molecule had an isoelectric point of 6.6 whereas the 210 kDa form of the horse ferritin molecule had isoelectric points of 5.1 and 4.9. Intact chicken liver ferritin particles were 13.4 +/- 0.8 nm (controlled-pore glass-purified) and 12.5 +/- 0.9 nm (affinity-purified) in diameter when viewed by electron microscopy. Horse spleen ferritin consisted of slightly smaller particles with an average diameter of 11.0 +/- 0.7 nm. However, ferritin from chicken liver and horse spleen co-migrated with an apparent molecular mass of 470 kDa when analysed by Sepharose 4B gel filtration chromatography. These results indicate that, consistent with results from other published purification methods, the chicken ferritin purified by the methods reported here exhibits both structural similarities to, and differences from, horse spleen ferritin.     

Thermotropic lateral translational motion of intramembrane particles in the inner mitochondrial membrane and its inhibition by artificial peripheral proteins

Freeze fracturing and deep etching have been used to study thermotropic lateral translational motion of intramembrane particles and membrane surface anionic groups in the inner mitochondrial membrane. When the inner membrane is equilibrated at low temperature, the fracture faces of both halves of the membrane reveal a lateral separation between intramembrane particles and particle free, large smooth patches. Such separation is completely reversed through free lateral translational diffusion by reversing the temperature. The low temperature induced, particle-free, smooth membrane patches appear to represent regions of protein-excluding, ordered bilayer lipid which form during thermotropic liquid crystalline to gel state phase transitions. When polycationic ferritin is electrostatically bound to anionic groups exposed at the membrane surface at concentrations which inhibit the activities of cytochrome c oxidase and succinate permease, the bound ferritin migrates with intramembrane particles during the thermotropic lateral separation between the membrane particles and smooth patches. When bound polycationic ferritin is cross-bridged with native ferritin, an artificial peripheral protein lattice forms in association with the surface anionic groups and diminishes the thermotropic lateral translational motion of intramembrane particles in the membrane. These results reveal that the anionic groups of metabolically active integral proteins which are known to be exposed at the surface of the inner mitochondrial membrane migrate with intramembrane particles in the plane of the membrane under conditions which induce lipid-protein lateral separations. In addition, cross-bridging of the anionic groups through an artificial peripheral protein lattice appears to diminish such induced lipid protein lateral separations.     

A biochemical comparison of normal human liver and hepatocellular carcinoma ferritins.

1. The iron contents, gel migration rates and isoelectric-focusing patterns of normal liver and hepatocellular carcinoma ferritins from the same patients were compared. 2. Sucrose-density-gradient centrifugation showed that the number of iron atoms per ferritin molecule was decreased to approximately half in carcinoma tissue when compared with normal liver. 3. On electrophoresis, hepatocellular carcinoma ferritin migrates faster and is therefore more negatively charged than normal liver ferritin, thus refuting the general view that the more negatively charged a ferritin molecule the greater its iron content. 4. Comparison of tumour and normal liver ferritin subunit compositions on acid/urea/polyacrylamide gels showed hepatocellular carcinoma ferritin to contain an additional, more negatively charged, subunit to normal liver ferritin. 5. Isoelectric focusing showed that hepatocellular carcinoma tissue contains isoferritins with isoelectric points intermediate between the ranges of normal liver and normal heart isoferritins.     

Possibly unique subunit(s) of human ferritins responsible for tissue specificity

The biochemical characteristics of human placental and hepatic ferritins were compared. By DEAE cellulose column chromatography, placental ferritin was found to contain more acidic isoferritins, while hepatic ferritin was richer in basic isoferritins. The electrofocusing patterns of subunits revealed that placental ferritin contained at least one unique subunit on the acidic side and might lack one subunit on the basic side as compared with hepatic ferritin. It is hypothesized that microheterogeneities, immunological as well as biochemical, existing between placental and hepatic ferritins cannot be explained merely in terms of different proportions of known acidic and basic subunits, but appear to have resulted from the existence of the unique acidic subunit of placental ferritin and possibly also a unique basic subunit of hepatic ferritin.     

Characterization of bovine serum ferritin-binding proteins

Ferritin-binding proteins (FBPs) in bovine serum were characterized by ferritin immunoassay, ferritin-binding activity, and immunoblotting. Serum ferritin, but not tissue ferritin, was precipitated by centrifugation at 14000 x g for 30 min, and bovine spleen ferritin added to bovine serum was precipitated by centrifugation at 1650 x g for 20 min. Two FBPs (FBP1 and FBP2) were purified from bovine serum by sequential chromatography on bovine spleen ferritin-Sepharose 4B affinity and Sephacryl S-300 columns. FBP1 separated into 82 kDa- and 26 kDa-bands on SDS-PAGE, while FBP2 separated into 55 kDa- and 26 kDa-bands. FBP1 and FBP2 were identified as IgM and IgG, respectively, by immunoblotting with alkaline phosphatase-labeled antibodies specific for bovine IgM, IgG, and IgA heavy chains. Given these results, we suggest that bovine FBPs are autoantibodies (IgM and IgG) to ferritin and that circulating ferritin exists as an immune complex.     

Upregulation of hemolymph and tissue ferritin by dietary HgCl2 in the wax moth Galleria mellonella

The effect of Hg treatment on hemolymph and tissue ferritin in the wax moth Galleria mellonella was examined by western blotting. At 48 h after feeding HgCl₂, the level of hemolymph ferritin increased approximately 1.8‐fold over that of control insects that were not fed HgCl₂, while there was a small increase in tissue ferritin. Time series experiments showed that tissue ferritin had a typically saturated pattern, with a maximum level from 24 to 72 h, although it decreased 12 h following HgCl₂ feeding, while hemolymph ferritin first decreased but subsequently increased. Tissue ferritin in the fat body, gut and Malpighian tubules, the main tissues of ferritin expression, was upregulated over time following treatment with Hg, and in particular, tissue ferritin in the gut increased by a large amount at 12–48 h. The results suggest that in G. mellonella, the ferritin‐inducible mechanisms following treatment with HgCl₂ are different for hemolymph and tissue ferritin, as are their biochemical properties.     

Ultrastructure of ferritin in the leaves of <Emphasis Type="Italic">Mesembryanthemum crystallinum</Emphasis> under stress conditions

The location and structure of ferritin in the parenchyma of leaf minor veins of the common ice plant (Mesembryanthemum crystallinum L.) treated with exogenous putrescine under salinity conditions were investigated by electron microscopy. Considerable aggregates of ferritin were detected in the chloroplasts of bundle sheath cells, in companion phloem cells, and other parenchyma cells of leaf minor veins. The structure of ferritin in the vascular parenchyma chloroplasts suggests that it was partially degraded and converted to phytosiderin. This point of view is based on indistinct structure of Fe-containing cores of ferritin molecules, break of distance between the cores, and their pronounced ability to aggregate and produce larger structures. Aggregation of Fe-containing cores apparently pointed to the destruction of ferritin protein envelope or its partial degradation. In a certain stage of ferritin destruction, electron-dense material and the structures resembling small vesicles appeared between the Fe-containing cores. Electron-dense inclusions, whose structure was similar to that of phytosiderin, were also detected in the vacuoles. Examination of the cross sections done without additional staining showed that the same as ferritin, phytosiderin in the chloroplasts and vacuoles was dark-colored against weakly colored cellular structures. In the vascular parenchyma of control plant leaves, the level of ferritin and phytosiderin was greater than in the mesophyll and much lower than in the plants simultaneously treated with NaCl and putrescine. In control material, iron cores of ferritin and phytosiderin were more light-colored and 2–3 times smaller in size than in the experimental treatment. Destruction of ferritin essentially did not occur in the mesophyll but was observed in the chloroplasts of bundle sheath cells on the border between the mesophyll and vascular bundle. The presence of much ferritin and phytosiderin on the border between the mesophyll and the vessels is accounted for by the fact that the vascular parenchyma is a buffer area that maintains a specific concentration of iron in the mesophyll of leaves and other parts of the plant. Within the cell, the role of such a buffer is performed by ferritin and vacuoles. Transformation of ferritin to insoluble hydrophobic phytosiderin is supposed to be an efficient way of withdrawing the excess of active iron from the cellular metabolism and therefore of relaxing oxidative stress. Ferritin and phytosiderin were detected not only in parenchyma cells of leaf minor veins but in sieve tubes as well. This suggests that iron may be transported within the plant as a component of protein complex.     

Glycoprotein Ib distribution on the surface of platelets in resting and activation states: An electron microscope study

Summary Using a monoclonal antibody (TM60) against glycoprotein (GP) Ib, we determined immunocytochemically how GPIb is distributed on the platelet surface. When glutaraldehyde-fixed platelets were incubated with TM60, a uniform distribution of ferritin particles which represent the localization of GPIb was observed on the surface membrane of platelets. The particles were distributed at intervals of about 100 nm. The number of ferritin particles on the surface of one side were 2070–4150 (2940 ± 790; mean ±s.d.,n = 10) under the scanning electron microscope. The distribution of ferritin particles was somewhat disarranged on the surface of unfixed platelets incubated with TM60 compared to that in the fixed platelets. Cluster-like structures of ferritin particles were observed in several places. When platelets were activated with ristocetin or thrombin, the distribution of ferritin particles was disturbed and cluster formation was observed in several places on the surface. These findings suggest that GPIb is uniformly distributed on the surface of platelets in the resting state, and that cluster formation occurs during activation of platelets.