title: Binding and endocytosis of apo- and holo-lactoferrin by isolated rat hepatocytes.
abstract: We characterized binding and endocytosis of 125I-bovine lactoferrin by isolated rat hepatocytes. Iron-depleted (apo-Lf), approximately 30% saturated (Lf), and iron-saturated (holo-Lf) lactoferrin were used. At 4 degrees C, cells bound 125I-apo-Lf and 125I-holo-Lf with nearly identical apparent first order kinetics (t1/2 = approximately 42 min). Holo-Lf and apo-Lf competed with each other for binding. Hepatocytes bound lactoferrin optimally at pH greater than or equal to 7 but poorly at pH less than or equal to 6. Ca2+ (greater than or equal to 100 microM) enhanced Lf binding to cells, and holo-Lf remained monomeric with Ca2+ present as determined by gel filtration chromatography. With Ca2+, cells exhibited approximately 10(6) high affinity sites (Kd approximately 20 nM) and approximately 10(7) low affinity sites (Kd approximately 700 nM) for both apo- and holo-Lf. Without Ca2+, cells bound 125I-holo-Lf by the low affinity component only. EGTA and dextran sulfate together released greater than or equal to 90% 125I-Lf prebound at 4 degrees C, but individually removed separate populations of surface-bound 125I-Lf. Cells bound 125I-Lf in a Ca(2+)-dependent manner with dextran sulfate present. We conclude that the high affinity but not the low affinity sites require Ca2+; only the low affinity sites are dextran sulfate-sensitive. Neither transferrin nor asialo-orosomucoid blocked lactoferrin binding to hepatocytes. Some cationic proteins but not others inhibited lactoferrin binding. At 37 degrees C, hepatocytes endocytosed 125I-apo-Lf and 125I-holo-Lf similarly, and hyperosmolality (greater than 500 mmol/kg) blocked uptake by approximately 90%. These data support the proposal that hepatocytes regulate blood lactoferrin concentration by receptor-mediated endocytosis.
authors.affiliation: Department of Biological Sciences, University of Notre Dame, Indiana 46556.
authors.forename: D D
authors.initials: DD
authors.lastname: McAbee
authors.forename: K
authors.initials: K
authors.lastname: Esbensen
mesh_terms: D000818:Animals; D001059:Apoproteins / Q000378:metabolism*; D001345:Autoradiography; D001667:Binding, Competitive; D002122:Calcium Chloride / Q000494:pharmacology; D002478:Cells, Cultured; D004533:Egtazic Acid / Q000494:pharmacology; D004705:Endocytosis; D007457:Iodine Radioisotopes; D007700:Kinetics; D007781:Lactoferrin / Q000378:metabolism*; D008099:Liver / Q000187:drug effects / Q000378:metabolism*; D015636:Magnesium Chloride / Q000494:pharmacology; D008297:Male; D009994:Osmolar Concentration; D051381:Rats; D011919:Rats, Inbred Strains; D011956:Receptors, Cell Surface / Q000302:isolation & purification / Q000378:metabolism*
chemical_list: D001059:Apoproteins; D007457:Iodine Radioisotopes; D011956:Receptors, Cell Surface; C039223:apolactoferrin; C038340:lactoferrin receptors; D015636:Magnesium Chloride; D004533:Egtazic Acid; D007781:Lactoferrin; D002122:Calcium Chloride