A reassessment of the assay for the asialoglycoprotein receptor and its use in the quantification of receptor distribution in hepatocytes

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Abstract
The assay for the fucose-binding protein described by Lehrman and Hill [(1983) Methods Enzymol. 98, 309-319] was adapted for the measurement of the asialoglycoprotein receptor in rat liver. The amount of ligand bound to the plasma-membrane-associated or affinity-purified receptor was acutely sensitive to the concentrations of Triton X-100 and NaCl in the assay; 0.02% (v/v) Triton X-100 increased ligand binding to the two preparations by 100% and 40%, respectively. Higher concentrations of detergent progressively decreased binding, and in 0.32% Triton X-100 it was about 30% of the value obtained in detergent-free buffer. The addition of increasing concentrations of NaCl to the assay progressively inhibited ligand binding to the membrane-associated receptor, whereas there was a 60% increase in binding to the pure receptor in the presence of 0.1-0.2 M-NaCl. These effects could not be identified in the original assay procedure described by Hudgin, Pricer, Ashwell, Stockert and Morell [(1974) J. Biol. Chem. 249, 5536-5543]. Using optimal assay conditions the binding of 125I-7b-D-galactosyl-bovine serum albumin to both the membrane-associated and purified receptor was inhibited by 50% by 1 nM-.beta.-D-galactosyl-bovine serum albumin and -asialoorosomucoid and by approx. 100 .mu.M-.beta.-L-fucosyl-bovine serum albumin, whereas .beta.-D-galactose, lactose and .beta.-L-fucose had no effect on ligand binding up to concentrations of 1 mM, 500 .mu.M and 5 mM, respectively. Kd values of 0.94 and 1.25 mM and Bmax values of 40 and 1660 pmol of D-galactosyl-bovine serum albumin bound/mg of receptor were obtained for the membrane-bound and purified receptor, respectively. Hill-plot analysis of the same data gave slopes of 0.96 and 1.01. Scatchard analysis of saturation-binding studies with other subcellular fractions indicated that the receptor was distributed in the proportions 72:23:2.5:2.5 between total microsomal fractions, plasma membrane, Golgi and canalicular membrane, respectively. The receptor was about 1% of the total protein in each compartment and was estimated to be about 0.3% of the total liver protein.