Abstract:
:We characterized endocytosis of iron-saturated (holo) and iron-depleted (apo) 125I-labeled bovine lactoferrin (Lf) by isolated rat hepatocytes. Hepatocytes ingested both Lf forms--determined by EGTA/dextran sulfate removal of surface-bound Lf--at maximal endocytic rates of 1.85 and 1.52 fmol cell-1 min-1 for 125I-apo-Lf and 125I-holo-Lf, respectively. First-order endocytic rate constants (37 degrees C) for 125I-apo-Lf and 125I-holo-Lf were 0.276 and 0.292 min-1, respectively. Regardless of Lf's iron content, hyperosmotic media (approximately 500 mmol/kg) inhibited Lf uptake by approximately 90%, indicating endocytosis of both Lf forms was primarily clathrin-dependent. Endocytosis of both Lf forms was not altered significantly in the presence of excess iron chelator desferrioxamine or rat holo-transferrin, or by cycloheximide treatment. Fluorescein isothiocyanate- and cyclohexanedione-modified Lf competed fully with native Lf for binding and endocytosis, indicating that, unlike human Lf, modification of lysine or arginine residues does not block the interaction of bovine Lf with cells. After binding Lf at 4 degrees C, cells at 37 degrees C internalized approximately 90% of Lf bound to Ca(2+)-dependent sites but not Lf bound to Ca(2+)-independent sites. Following uptake, hepatocytes released acid-soluble (degraded) products of 125I-Lf biphasically at 37 degrees C, an initial rapid phase within the first 20 min--more pronounced with 125I-holo-Lf--followed by a sustained linear release of 298 and 355 molecule equiv cell-1 min-1 for 125I-apo-Lf and 125I-holo-Lf, respectively. At 4 degrees C, both digitonin-permeabilized and intact cells bound approximately 1.1 x 10(6) 125I-Lf molecules to Ca(2+)-dependent sites per cell, indicating that hepatocytes do not contain a sizeable intracellular pool of these sites. Moreover, cells retained > 70% of Ca(2+)-dependent sites on the surface during sustained Lf endocytosis. Thus, these Lf binding sites recycle during endocytosis at an estimated 4-5 min/circuit.
journal_name
Biochemistryjournal_title
Biochemistryauthors
McAbee DD,Nowatzke W,Oehler C,Sitaram M,Sbaschnig E,Opferman JT,Carr J,Esbensen Kdoi
10.1021/bi00212a046subject
Has Abstractpub_date
1993-12-14 00:00:00pages
13749-60issue
49eissn
0006-2960issn
1520-4995journal_volume
32pub_type
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