[Uptake and intracellular metabolism of ferritin by primary cultured rat hepatocytes].
The uptake and intracellular metabolism of radiolabeled ferritin in rat hepatocytes were investigated. A receptor assay performed by incubating isolated hepatocytes with 125I-labeled ferritin at 37 degrees C for 1 h revealed 37,000 ferritin binding sites/cell with an affinity constant (Ka) of 1.8 X 10(9) M-1. The hepatocytes preincubated with 125I-labeled ferritin at 37 degrees C for 1 h were further incubated in a ligand-free medium at 37 degrees C for 4 h, and then the distribution of the label in the subcellular fractionations of hepatocytes was analyzed by 30% Percoll density gradient centrifugation. The label was transported from the plasma membrane fraction to the lysosome/mitochondria fraction and the cytosol fraction. Meanwhile, both of trichloroacetic acid (TCA)-soluble and TCA-precipitable 125I in the incubation medium increased with the incubation time. In order to study the intracellular metabolism of endocytosed ferritin, the hepatocytes preloaded with 59Fe-125I-double labeled ferritin were incubated at 37 degrees C for 4 h in the ligand-free medium containing 100 mumol/l chloroquine (a lysosomal metabolic inhibitor) or 10 mmol/l sodium cyanide (a mitochondrial metabolic inhibitor). Chloroquine increased TCA-precipitable 125I in the medium with a corresponding decrease in TCA-soluble 125I. However, no effect with sodium cyanide was observed. Radioactivities in the incubation medium were then divided into two components by HPLC gel filtration. One had a molecular weight similar to intact ferritin, 98% of which reacted to an anti-rat liver ferritin antiserum, while the other had a molecular weight of less than 1,000, 25% of which reacted to the antiserum. Chloroquine increased the amounts of intact ferritin released into the medium and decreased the degraded fragments. These results suggest two pathways of the endocytosed ferritin in hepatocytes; one is a degradation of ferritin in lysosome, which results in a storage of iron in the cells and an excretion of degraded peptides outside the cells, and the other is exocytosis of intact ferritin from the cells by a similar mechanism to "diacytosis" proposed for asialoglycoprotein.
Mikami
T
T
Second Department of Biochemistry, Nippon Medical School, Japan.
D000818:Animals; D001665:Binding Sites; D002478:Cells, Cultured; D004705:Endocytosis / Q000502:physiology*; D005293:Ferritins / Q000378:metabolism*; D033862:Iron-Binding Proteins; D008099:Liver / Q000166:cytology / Q000378:metabolism*; D008247:Lysosomes / Q000378:metabolism; D008297:Male; D051381:Rats; D011919:Rats, Inbred Strains; D011956:Receptors, Cell Surface / Q000502:physiology
D033862:Iron-Binding Proteins; D011956:Receptors, Cell Surface; C027293:ferritin receptor; D005293:Ferritins