Ferritin iron kinetics and protein turnover in K562 cells.
The binding, incorporation, and release of iron by ferritin were investigated in K562 cells using both pulse-chase and long term decay studies with 59Fe-transferrin as the labeled iron source. After a 20-min pulse of labeled transferrin, 60% of the 59Fe was bound by ferritin with the proportion increasing to 70% by 4 h. This initial binding was reduced to 35% when the cells were exposed to the chelator desferrioxamine (5 mM) for an additional 30 min. By 4 h the association of 59Fe with ferritin was unaffected by the presence of the chelator, and levels of 59Fe-ferritin were identical to those in control cells (70%). Between 4-10h there was a parallel decline in 59Fe-ferritin in both control and desferrioxamine-treated cells. When incoming iron was bound by ferritin it was, therefore, initially chelatable but with time progressed to a further, nonchelatable compartment. In turnover studies where ferritin was preloaded with 59Fe by overnight incubation, 50% of the label was released from the protein by 18 h, contrasting with a t 1/2 for cellular iron release of approximately 70 h. The half-time of 59Fe release from ferritin was accelerated to 11 h by the presence of desferrioxamine. The half-time for ferritin protein turnover determined by [35S]methionine labeling was approximately 12 h in the presence or absence of the chelator. Thus, when the reassociation of iron with ferritin was prevented by the exogenous chelator there was a concordant decay of both protein and iron moieties. The direct involvement of lysosomes in this turnover was demonstrated by the use of the inhibitors leupeptin and methylamine which stabilized both 59Fe (t 1/2 = 24 h) and 35S (t 1/2 = 25.6 h) labels. We conclude that in this cell type the predominant mechanism by which iron is released from ferritin is through the constitutive degradation of the protein by lysosomes.
Roberts
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S
Liver Unit, King's College School of Medicine and Dentistry, London, United Kingdom.
Bomford
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A
D000818:Animals; D002460:Cell Line; D003676:Deferoxamine / Q000494:pharmacology; D004591:Electrophoresis, Polyacrylamide Gel; D005293:Ferritins / Q000378:metabolism*; D007501:Iron / Q000378:metabolism*; D007700:Kinetics; D007976:Leupeptins / Q000494:pharmacology; D004369:Pentetic Acid / Q000494:pharmacology; D011506:Proteins / Q000378:metabolism*
D007976:Leupeptins; D011506:Proteins; D004369:Pentetic Acid; D005293:Ferritins; D007501:Iron; D003676:Deferoxamine; C032854:leupeptin