Biomedical subjects
C van der Heul
Publications and source records attributed to C van der Heul.
Analysis of iron-binding components in the low molecular weight fraction of rat reticulocyte cytosol.
Rat reticulocytes were incubated with rat 125I-Tf-59Fe under conditions inhibiting heme synthesis. Cytosol, prepared from the reticulocytes, was separated and analysed by gel filtration and Amicon Ultrafiltration. An iron-containing low molecular weight fraction derived from the cytosol was further analysed by HPLC size-exclusion chromatography and HPLC reversed phase chromatography. Conditions inhibiting heme synthesis and uncoupling the oxidative phosphorylations lead to a large increase in the Fe-containing low molecular weight fraction in the cytosol. The components in the low molecular weight fraction have an apparent molecular weight of 5500 Dalton as determined with HPLC size-exclusion chromatography. The low molecular weight fraction contained several iron chelating components like glycin, 1/2 cystine and citrate, but no specific iron-binding proteins, nucleotides or pyrophosphate.
Sialic acid-deficient serum and cerebrospinal fluid transferrin in a newly recognized genetic syndrome.
Identical twin-sisters with evidence of a demyelinating disease showed multiple serum glycoprotein abnormalities. The association of a low serum iron level and a normal blood haemoglobin suggested an abnormality of transferrin too. This was confirmed by finding a sialic acid-deficiency of this glycoprotein in serum as well as in cerebrospinal fluid.
A simple method for the separation and quantitation of neutral carbohydrates of glycoproteins in the one nanomole range on an adapted amino acid analyzer.
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Are lysosomes directly involved in the iron uptake by reticulocytes?
The mechanism by which iron is transferred from the plasma protein transferrin into erythroid precursors for incorporation in heme is not completely understood. To show a direct functional role of lysosomes in the process of iron uptake we tried to isolate lysosomes from reticulocytes, which have been incubated with 125ITf59Fe. However, with various cell fractionating techniques described for liver cells no pure lysosomes from reticulocytes could be obtained. Fluorescence and electron microscopy showed that reticulocytes hardly contain well-defined lysosomes. There are several indications that in reticulocytes acid vacuoles instead of lysosomes are involved in the removal of iron from endocytosed transferrin. The presence of apoTf, monoferric TfFe(A), monoferric TfFe(B) in the medium after incubation of reticulocytes with diferric transferrin, together with the fact that both iron binding sites of transferrin release their iron at pH present in acid vacuoles, suggests a second mechanism of iron uptake by reticulocytes, in which acid vacuoles are not involved.
Two mechanisms are involved in the process of iron-uptake by rat reticulocytes.
The iron uptake by red cell precursors has been studied in the presence of the carboxylic ionophore monensin, which achieves a concentration dependent inhibition of iron uptake, without influencing the transferrin uptake. It seems that two mechanisms are involved: Iron is released from endocytosed transferrin by acid vesicles. Iron is released from surface-receptor-bound transferrin at the plasma membrane, without internalization of the transferrin receptor complex.
Iron uptake from sialo transferrins by rat reticulocytes.
Preparative isoelectric focusing of human diferric transferrin preparations yielded seven bands with different isoelectric points, due to differences in sialic acid content. Incubation of rat reticulocytes at 37 and 4 degrees C with differic preparations of four of these transferrin forms labeled with 59Fe and 125I show no differences in membrane binding of iron and transferrin and in iron uptake. Hence it is concluded that the carbohydrate chains are not directly involved in the process of iron delivery to reticulocytes.
In vitro and in vivo studies of iron delivery by human monoferric transferrins.
According to the Fletcher-Huehns hypothesis there exists a functional difference between the two iron-binding sites of transferrin. In this study we present the results of an evaluation of this hypothesis in vitro and in vivo with human pure monoferric transferrins obtained by preparative isoelectric focusing in granulated gels. The uptake of iron from monoferric transferrins TfFeC and FeNTf by erythroid bone marrow cells, hepatocytes and stimulated T-lymphocytes in vitro was equal, even when both monoferric transferrins were present together in the incubation medium. Ferrokinetic studies in vivo, performed with both pure monoferric transferrins, showed that transferrin TfFeC, as well as transferrin FeNTf, mainly deliver their iron to the erythron. As red cell 59Fe utilization, red cell iron turnover and other ferrokinetic parameters, obtained from this study, were identical too it is evident that both iron-binding sites of transferrin are functionally homogeneous in vivo, with respect to iron delivery.
The microheterogeneity of human transferrins in biological fluids.
The serum, cerebrospinal fluid, amniotic fluid and synovial fluid transferrins have been examined by isoelectric focusing in a pH gradient from 5-7. In all biological fluids the presence of transferrins with a varying content of sialic acid has been shown. Differences between the fluids with regard to transferrin have been noticed.
Microheterogeneity of human serum transferrins: a consequence for immunochemical determinations?
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The heterogeneity of human serum transferrin and human transferrin preparations on isoelectric focusing gels; no functional difference of the fractions in vitro.
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Characterization and localization of the transferrin receptor on rat reticulocytes.
1. A further characterization and localization of the membrane receptor for transferrin on rat reticulocytes is described. PAGE studies with a purified membrane complex B2, from which the functional role in transferrin binding and iron uptake has been shown previously, showed that the transferrin receptor is localized on a membrane protein with a mol. wt of approximately 70-80.10(3). 2. Selective solubilization of the rat reticulocyte membrane has shown that this receptor protein belongs to one of the minor integral membrane polypeptides, embedded in the lipid bilayer of the membrane. 3. Proteolipid complexes, glycolipids and sialoglycoproteins of the rat reticulocyte membrane play no direct role in the binding capacity of the receptor.
Plasma infusion and plasma exchange in chronic idiopathic polyneuropathy.
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Observations on the iron status during pregnancy in rats. Iron transport from mother to fetus.
The objective of the present study was to investigate a transfer of liver iron in rats from mother to fetus during pregnancy. A decrease in maternal liver iron and an increase in fetal liver iron, in groups of 10 rats, have been shown, based on iron analysis and specific activity of 59Fe.
Isolation of the two monoferric human transferrins by preparative isoelectric focussing.
1. Human transferrin was labelled with Fe(III) in nitrilo triacetate buffer at one of its two metal binding sites by variation of the pH. The A-site was preferentially labelled at pH 7.2, the B-site at pH 9.2. 2. The monoferric transferrin preparations were subjected to preparative isoelectric focussing, isolated from the granulated gels, and checked for homogeneity and purity on an analytical scale by isoelectric focussing. 3. The technique for obtaining stable monoferric transferrin (Fe)(A) and transferrin (Fe)(B) preparations is described in detail.
[Various aspects of iron metabolism].
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Binding sites of iron transferrin on rat reticulocytes. Inhibition by specific antibodies.
1. In the process of iron uptake by precursors of the erythrocytes probably more than one membrane component is involved; besides the specific transferrin receptor, another membrane component with a high iron activity after incubation with 59Fe can be isolated. 2. A striking resemblance exists between rat and human reticulocyte components which are involved in the process of iron uptake. 3. Incubation of reticulocytes with Fab fragments of an antibody against the membrane receptor for transferrin causes a concentration-dependent decrease in transferrin binding and iron uptake. 4. The membrane receptor complex isolated is still heterogeneous; analytical ultracentrifugation studies suggest a molecular weight lower than 230,000. 5. Intact immature red cells are necessary for specific binding of transferrin with the receptor followed by iron uptake. This is the only mechanism for iron uptake. 6. Immunofluorescence studies showed that the receptor for transferrin is localized at the outside of the cell membrane.
Analysis of the iron-binding sites of transferrin by isoelectric focussing.
1. Human transferrin was labelled with ferric nitrilotriacetate (FeNTA) at one of its two metal binding sites by variation of the pH. 2. Four transferrin forms, transferrin, transferrin(Fe) (A-site), transferrin(Fe) (B-site) and transferrin(2Fe) could be separated on flat bed gels by isoelectric focussing. 3. Incubation time, temperature and medium play an important role in the specificity of the binding of Fe. In NTA-pH-buffer, at 0.5 Fe-saturation, the A-site was preferentially labelled at pH 7--8, the B-site at a pH 8--9. 4. Under physiological conditions iron from the B-site has the tendency to move to the A-site.