High resolution separation of metalloproteins and other iron compounds based on native gel electrophoresis followed by 59Fe autoradiography is described. Lysates of mouse spleen erythroid cells metabolically labeled with 59Fe-transferrin were separated on 3-20% polyacrylamide gradient gels in the presence of Triton X100 and detected by autoradiography. In addition to ferritin and hemoglobin, several compounds characterized by their binding of iron under different conditions were described. Iron chelatable by desferrioxamine migrated in the region where several high-molecular weight compounds were detected by silver staining. The technique is nondissociative, allowing identification of iron compounds with the use of specific antibodies. Cellular iron transport and the action of iron chelators on specific cellular targets can be investigated in many small biological samples in parallel.

• MeSH
• autoradiografie MeSH
• chelátory železa chemie   MeSH
• deferoxamin chemie   MeSH
• elektroforéza v polyakrylamidovém gelu MeSH
• myši inbrední BALB C MeSH
• myši MeSH
• senzitivita a specificita MeSH
• ultrafiltrace MeSH
• železo chemie   izolace a purifikace   MeSH
• zvířata MeSH
• Check Tag
• mužské pohlaví MeSH
• myši MeSH
• zvířata MeSH
• Publikační typ
• práce podpořená grantem MeSH

Succinylacetone (SA) is an inhibitor of heme synthesis that acts on the enzyme delta-aminolevulinic acid dehydratase. When reticulocytes are incubated with 59Fe-transferrin (59Fe-Tf) in the presence of SA, there is an accumulation of 59Fe in the mitochondrion and in a cytosolic non-heme intermediate that has been described as a putative Fe transporter (Adams et al, Biochim Biophys Acta 1012:243, 1989). Considering these observations, the present study was designed to examine the intermediates of Fe metabolism in control and SA-treated reticulocytes. This investigation showed that in the cytosol of control cells, most 59Fe was incorporated into hemoglobin (Hb) with a minor amount entering ferritin. In addition, a previously unrecognized cytosolic intermediate was identified (band X) that was absent when heme synthesis was inhibited with SA. Upon reincubation of SA-treated reticulocytes with protoporphyrin IX, band X initially increased in intensity and then decreased later in the incubation. In contrast, when 59Fe-labeled control cells were reincubated in the presence of SA and unlabeled diferric Tf, there was a marked decrease in the intensity of band X. These experiments suggest that component X may be an intermediate involved in the transfer of heme in the cytosol. Alternatively, these data could also be interpreted as indicating that band X may be a short-lived hemoprotein. We have confirmed the presence of an 59Fe-containing molecule in the cytosol of SA-treated reticulocytes (band Y) that is not present in control cells. However, when cells were incubated with 59Fe-Tf plus SA and then chased in the presence of SA and unlabeled diferric Tf, there was no decrease in this cytosolic pool of Fe, suggesting that it was not a intermediate supplying Fe for either ferritin or heme synthesis. Finally, there is little low molecular weight (Mr) Fe in reticulocytes, and our studies suggest that the low-Mr Fe present does not behave as an intermediate. Moreover, after inhibition of heme synthesis with SA, 59Fe in the low-Mr compartment was markedly decreased, suggesting that this component may be heme or a low-Mr heme-containing molecule. Considering the apparent lack of a cytosolic Fe transporter in rabbit reticulocytes, an alternative model of intracellular Fe transport is proposed that does not implicate a potentially toxic intermediate pool of low-Mr Fe complexes.

• MeSH
• biologické modely MeSH
• chelátory železa farmakologie   MeSH
• cytosol metabolismus   MeSH
• deferoxamin farmakologie   MeSH
• hem biosyntéza   MeSH
• hemoglobiny biosyntéza   MeSH
• heptanoáty farmakologie   MeSH
• inhibitory enzymů farmakologie   MeSH
• kompartmentace buňky MeSH
• králíci MeSH
• krevní proteiny metabolismus   MeSH
• membránové proteiny metabolismus   MeSH
• mitochondrie metabolismus   MeSH
• porfobilinogensynthasa antagonisté a inhibitory   metabolismus   MeSH
• protoporfyriny metabolismus   MeSH
• retikulocyty chemie   účinky léků   ultrastruktura   MeSH
• transferin metabolismus   MeSH
• železo analýza   metabolismus   MeSH
• zvířata MeSH
• Check Tag
• králíci MeSH
• zvířata MeSH
• Publikační typ
• práce podpořená grantem MeSH

• MeSH
• akutní erytroblastická leukemie MeSH
• buněčná diferenciace MeSH
• erytroblasty patologie   MeSH
• myši MeSH
• proteiny vázající RNA MeSH
• techniky in vitro MeSH
• techniky tkáňových kultur MeSH
• železo metabolismus   MeSH
• zvířata MeSH
• Check Tag
• myši MeSH
• zvířata MeSH

Cílem je popsat na molekulární úrovni řízení vstupu železa z transferinu do erytroidních buněk. Prozkoumat souvislost mezi syntézou hemu a aktivitou regulačního proteinu, který mění stabilitu mRNA pro transferinový receptor. XXX XXX XXX

• MeSH
• receptory transferinu MeSH

• Konspekt
• Patologie. Klinická medicína
• NLK Obory
• hematologie a transfuzní lékařství
• NLK Publikační typ
• závěrečné zprávy o řešení grantu IGA MZ ČR