Isolation and Characterization of Iron‐Binding Proteins from Rat Intestinal Mucosa

Huebers, Helmut A.; Huebers, Eiko; Rummel, W.; Crichton, Robert R.

doi:10.1111/j.1432-1033.1976.tb10569.x

Cited by 107 publications

(58 citation statements)

References 21 publications

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“…Rat serum transferrin was purified by a modification of the method in [7] using (NH&SO4 precipitation, DEAE-cellulose chromatography, chromatography on Sepharose-protein A and a final chromatography on CM-celhrlose [6]. Transferrin was labelled with 'H by reductive methylation [8] using sodium ['Hlborohydride and the apotransferrin was labelled with 59Fe as ferric citrate in order to have a specific activity close to that for 3H 161.…”

Section: Methodsmentioning

confidence: 99%

Transferrin protein and iron uptake by cultured rat fibroblasts

et al. 1979

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Section: Methodsmentioning

confidence: 99%

Transferrin protein and iron uptake by cultured rat fibroblasts

et al. 1979

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“…Rat liver ferritin was isolated as described in [22]. The homogeneity of the preparation was established by polyacrylamide gel electrophoresis in sodium dodecyl sulfate [23] and by amino acid analysis after hydrolysis with 6 M HCI for 16 h at 110°C using a Locarte amino acid analyser (Locarte Co., London, England).…”

Section: Purification Of Ferritinmentioning

confidence: 99%

Transferrin Uptake by Cultured Rat Embryo Fibroblasts

Octave¹,

Schneider²,

Crichton³

et al. 2005

European Journal of Biochemistry

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The uptake of doubly labelled (5yFe and 3H] rat serum transferrin by rat embryo fibroblasts in culture has been studied. 5yFe is taken up by the cells in a process dependent on time and temperature which, at 37 C, is almost saturable with transferrin concentration. The iron is incorporated in part into ferritin. In contrast, the uptake of 3H label at 37°C is much less than that of 59Fe and is associated with a saturable uptake of demonstrably intact transferrin and with the time-dependent and concentration-dependent release of transferrin degradation products from the cells. Low temperature binding studies indicate that 60-100 x lo3 transferrin molecules are bound to high-affinity specific plasma-membrane receptors ( K a : 1.1 x lo7 M-'), and that a large number of low-affinity sites also exist. Transferrin bound to low-affinity sites can be readily released from the cells by washing or by reincubation in fresh medium.Short-term kinetic experiments at 37 "C imply that a mechanism exists whereby iron is rapidly taken up from transferrin and the iron-depleted transferrin is released intact to the extracellular medium. After 4 min (I-min incubation at 37 "C, 1-min washing and 2-min reincubation) half of the [3H]transferrin is released into the medium while almost all of the 5yFe remains associated with the cells.On the basis of our results we suggest that iron uptake from transferrin involves both receptor-mediated and flluid-phase endocytosis. In the former, the fusion of the endocytic vacuole with a lysosome would provide a favourable environment for iron release ; the iron-depleted transferrin would then be returned to the extracellular medium during the recycling of the membrane. In the latter, after the pinocytic vacuole gains access to lysosomes the iron would be released and the protein digested. In both mechanisms the iron would pass into the cytosol and be incorporated into ferritin or be used for mitochondria1 haem synthesis.

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“…The specific activities obtained ranged from 3 x lo5 dis min-' pg protein-' to 7x105dismin-' pgprotein-'for 3Handfrom104dismin-' pg-' protein-' to 2 x lo4 dis min-' kg protein-' for 59Fe. Ferritin was purified from rat liver as in [23] and antiferritin and antitransferrin antibodies were obtained in rabbits using the immunization procedure as described in [24]. Triton WR 1339 was purchased from Ruger Chemical Co Inc., (Irvington, NJ, USA) and digitonin from E. Merck (Darmstadt, FRG).…”

Section: Ma Teriulsmentioning

confidence: 99%

Subcellular localization of transferrin protein and iron in the perfused rat liver

Sibille

Octave

Schneider

et al. 1986

European Journal of Biochemistry

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The subcellular localization of 3H-labelled 59Fe-loaded transferrin accumulated by the liver has been studied by means of cell fractionation techniques.More than 96% of the s9Fe present in the liver of rats perfused with 59Fe-labelled transferrin is recovered in the parenchymal cells.Rat livers were perfused with 10 pg/ml 3H-labelled 59Fe-saturated transferrin, homogenized separated in nuclear (N), mitochondrial (M), light mitochondrial (L), microsomal (P) and supernatant ( S ) fractions; M, L and P fractions were further analysed by isopycnic centrifugation in sucrose gradients. 3H label distributes essentially around densities of 1.13 -1.14 g/ml overlapping to a large extent with the distribution of galactosyltransferase, the marker enzyme of the Golgi complex.However, after treatment with low concentrations of digitonin the 'H label dissociates from galactosyltransferase and is shifted to higher densities, suggesting an association of transferrin with cholesterolrich endocytic vesicles which could derive from the plasma membrane.59Fe is mostly found in the supernatant fraction largely in the form of ferritin, as indicated by its reaction with antiferritin antibodies. In the mitochondrial fraction the density distribution of 5yFe suggests an association with lysosomes and/or mitochondria. In contrast to the lysosomal enzyme cathepsin B, the density distribution of "Fe was only slightly affected by pretreatment of the rats with Triton WR 1339, suggesting its association with the mitochondria. At 15 "C, 59Fe and 3H labels are recovered together in low-density endocytic vesicles.On the basis of our results we suggest that, at low extracellular transferrin concentration, iron uptake by the liver involves endocytosis of the transferrin protein. The complex is interiorized in low-density acidic vesicles where iron is released. The iron passes into the cytosol, where it is incorporated into ferritin and into the mitochondria. The iron-depleted transferrin molecule would then be returned to the extracellular medium during the recycling of the plasma membrane.

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Isolation and Characterization of Iron‐Binding Proteins from Rat Intestinal Mucosa

Cited by 107 publications

References 21 publications

Transferrin protein and iron uptake by cultured rat fibroblasts

Transferrin protein and iron uptake by cultured rat fibroblasts

Transferrin Uptake by Cultured Rat Embryo Fibroblasts

Subcellular localization of transferrin protein and iron in the perfused rat liver

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