IDENTIFICATION AND ISOLATION OF A 45-KDA CALCIUM-DEPENDENT LACTOFERRIN RECEPTOR FROM RAT HEPATOCYTES

Isolated rat hepatocyes bind, internalize, and degrade bovine lactofer rin (Lf) via high-affinity Ca2+-dependent sites [<10(6) sites/cell; Mc Abee et al., (1993) Biochemistry 32, 13749-13760]. In this study, we i dentified a 45-kDa Ca2+-dependent Lf binding protein on rat hepatocyte s by three independent approaches. First, dithiobis(sulfosuccimidylpro prionate) (DTSSP) cross-linked I-125-Lf to a 45-kDa adduct in a Ca2+-d ependent manner on intact cells. The I-125-labeled cross-linked comple xes were absent when either surface-bound I-125-Lf was stripped prior to cross-linking or an excess of unlabeled Lf was included in the DTSS P reaction. Second, I-125-Lf bound to a 45-kDa hepatocyte polypeptide in a Ca2+-dependent fashion following incubation with SDS-PAGE fractio ned hepatocyte membrane proteins absorbed on nitrocellulose. Third, wh en Triton X-100 extracts of hepatocyte membrane ghosts were chromatogr aphed on Lf-agarose, a 45-kDa polypeptide (p45) was eluted by EGTA. Co lumn fractions enriched in p45-but not those depleted of p45-possessed soluble Lf receptor activity as determined by competition binding ass ay. Monospecific polyclonal anti-p45 IgG detected p45 in crude hepatoc yte ghost homogenates and blocked vigorously I-125-Lf binding and endo cytosis to intact rat hepatocytes. We conclude, therefore, that p45 co nstitutes the Ca2+-dependent Lf receptor on isolated rat hepatocytes.