RESUMO
1. Endosomes were isolated from K 562 cells after 3 min after the endocytosis of a single cohort of transferrin molecules. 2. The change in 125I/59Fe ratio of heavy and light endosomes, relative to that of the transferrin used and 59Fe from light endosomes. 3. Incubation of heavy and light endosomes with PBS or PIH showed equal ATP specific iron release from both heavy and light endosomes, but in the presence of a NADH/NAD+ redox couple iron release from light endosomes was reduced. 4. Incubation of heavy and light endosomes with PIH and NEM did not completely abolish ATP specific iron release from heavy and light endosomes.
Assuntos
Endocitose , Ferro/metabolismo , Etilmaleimida/farmacologia , Humanos , Quelantes de Ferro/farmacologia , Isoniazida/análogos & derivados , Isoniazida/farmacologia , Lactatos/farmacologia , Fenantrolinas/farmacologia , Proteínas/análise , Piridoxal/análogos & derivados , Piridoxal/farmacologia , Transferrina/metabolismo , Células Tumorais Cultivadas , Desacopladores/farmacologiaRESUMO
Transferrin bound to K 562 cells at 4 degrees C was internalized quickly on temperature shift to 37 degrees C. Endosomes were isolated according to two different procedures. The endosome fraction was shown to be heterogeneous and consisted of two vesicle populations, differing in density properties and iron content. Iron was partially released from endosomes to the supernatant after 3 and 5 min endocytosis. Isolated endosomes, still capable of internal acidification, did not release iron on incubation with ATP. However, endosomes did release iron on incubation with the iron chelator pyridoxal-isonicotinoyl hydrazone. Gel-filtration of solubilized endosomes demonstrated the presence of the transferrin-transferrin receptor complexes, free transferrin and free low molecular weight iron.
Assuntos
Endocitose , Endossomos/metabolismo , Ferro/metabolismo , Transferrina/metabolismo , Trifosfato de Adenosina/farmacologia , Fracionamento Celular , Linhagem Celular , Centrifugação , Endossomos/efeitos dos fármacos , Humanos , Quelantes de Ferro , Radioisótopos de Ferro , Isoniazida/análogos & derivados , Isoniazida/farmacologia , Leucemia Eritroblástica Aguda , Piridoxal/análogos & derivados , Piridoxal/farmacologiaRESUMO
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.
