Abstract
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.
Published Version
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