Identification and isolation of a 45-kDa calcium-dependent lactoferrin receptor from rat hepatocytes.
Isolated rat hepatocytes bind, internalize, and degrade bovine lactoferrin (Lf) via high-affinity Ca2+-dependent sites [<10(6) sites/cell; McAbee et al., (1993) Biochemistry 32, 13749-13760]. In this study, we identified a 45-kDa Ca2+-dependent Lf binding protein on rat hepatocytes by three independent approaches. First, dithiobis(sulfosuccimidylproprionate) (DTSSP) cross-linked 125I-Lf to a 45-kDa adduct in a Ca2+-dependent manner on intact cells. The 125I-labeled cross-linked complexes were absent when either surface-bound 125I-Lf was stripped prior to cross-linking or an excess of unlabeled Lf was included in the DTSSP reaction. Second, 125I-Lf bound to a 45-kDa hepatocyte polypeptide in a Ca2+-dependent fashion following incubation with SDS-PAGE fractioned hepatocyte membrane proteins absorbed on nitrocellulose. Third, when Triton X-100 extracts of hepatocyte membrane ghosts were chromatographed on Lf-agarose, a 45-kDa polypeptide (p45) was eluted by EGTA. Column fractions enriched in p45--but not those depleted of p45--possessed soluble Lf receptor activity as determined by competition binding assay. Monospecific polyclonal anti-p45 IgG detected p45 in crude hepatocyte ghost homogenates and blocked vigorously 125I-Lf binding and endocytosis to intact rat hepatocytes. We conclude, therefore, that p45 constitutes the Ca2+-dependent Lf receptor on isolated rat hepatocytes.
Bennatt
D J
DJ
Department of Biological Sciences, University of Notre Dame, Notre Dame, Indiana 46556, USA.
McAbee
D D
DD
D000818:Animals; D015153:Blotting, Western; D002118:Calcium / Q000494:pharmacology*; D002462:Cell Membrane / Q000737:chemistry; D002846:Chromatography, Affinity; D003432:Cross-Linking Reagents; D004533:Egtazic Acid; D004591:Electrophoresis, Polyacrylamide Gel; D005293:Ferritins / Q000378:metabolism; D006801:Humans; D007457:Iodine Radioisotopes; D007781:Lactoferrin / Q000378:metabolism; D008099:Liver / Q000737:chemistry*; D008297:Male; D008970:Molecular Weight; D017830:Octoxynol; D051381:Rats; D017207:Rats, Sprague-Dawley; D011956:Receptors, Cell Surface / Q000302:isolation & purification* / Q000378:metabolism
D003432:Cross-Linking Reagents; D007457:Iodine Radioisotopes; D011956:Receptors, Cell Surface; C038340:lactoferrin receptors; D004533:Egtazic Acid; D017830:Octoxynol; D005293:Ferritins; D007781:Lactoferrin; D002118:Calcium