Different modes of ligand binding to the hepatic galactose/N-acetylgalactosamine lectin on the surface of rabbit hepatocytes

Abstract

A study of the binding of three different 125I-labeled, galactose-terminated ligands to the hepatic galactose/N-acetylgalactosamine-specific lectin found on the surface of rabbit hepatocytes revealed that the different ligands manifest different physical parameters of binding. Asialoorosomucoid (125I-ASOR) binding was best described as involving two independent classes of binding sites on rabbit hepatocytes, with 161 000 sites/cell with a dissociation constant of 0.44 nM and 292 000 sites/cell with a Kd of 9.7 nM. Asialotriantennary glycopeptide purified from human alpha-1 protease inhibitor and modified with tyrosine at the N-terminus to permit radioiodination (TRI) [Lee, Y. C., Townsend, R. R., Hardy, M. R., Lönngren, J., Arnarp, J., Haraldsson, M., & Lönn, H. (1983) J. Biol. Chem. 258, 199-202] was also found to bind to two apparent classes of binding sites but with different binding parameters: 292 000 sites/cell of Kd = 1.47 nM and 982 000 sites/cell of Kd = 25.3 nM. A synthetic ligand, alpha,beta-diaspartamide of tris[(beta-lactosyloxy)methyl](6-aminohexanamido)methane (di-tris-lac) containing six nonreducing galactose residues [Lee, R. T., Lin, P., & Lee, Y. C. (1984) Biochemistry 23, 4255-4261], was found to bind to 817 000 sites/cell of Kd = 0.63 nM and 1.23 X 10(6) sites/cell of Kd = 25.3 nM. Thus, there were many more total binding sites for TRI or di-tris-lac on the surface of rabbit hepatocytes than there were for asialoorosomucoid, although the dissociation constants were similar for all three ligands.(ABSTRACT TRUNCATED AT 250 WORDS)

Glycine Dimerization Motif in the N-terminal Transmembrane Domain of the High Density Lipoprotein Receptor SR-BI Required for Normal Receptor Oligomerization and Lipid Transport

“…Instead, if identical binding site are near one another in a "lattice," either as adjacent monomers or oligomers, the initial binding of a relatively large ligand to one site could, without altering the structure or intrinsic binding properties of neighboring sites, sterically interfere with the access of a second ligand to one of the neighboring binding sites (81)(82)(83)(84). This initial binding effectively increases the K D of the neighboring unbound sites, resulting in negative cooperativity (85)(86)(87). SR-BI forms clusters on the surfaces of ldlA[mSR-BI] and other cells (70,88), and it forms oligomers (Refs.…”

Sequence of a second human asialoglycoprotein receptor: conservation of two receptor genes during evolution.

“…First, both HI and H2 (or Ri and R2) could be present at the cell surface but have different ligand-binding properties. The ASGP receptors on the surface of rabbit hepatocytes exhibit two affinities for single ligands (29), but it is not known how many ASGP receptors exist in rabbit hepatocytes. Second, only one of the two ASGP receptors could be present on the cell surface.…”

Enhancement of galactose/N-acetylgalactosamine receptor activity on the surface of freshly isolated rat hepatocytes: evidence for masking of receptor sites by inhibitors derived from collagenase preparations.

“…Removal of the collagenase inhibitor from the hepatocyte surface by these various treatments appeared not only to affect the receptor site number but also to alter the affinity constants (within a range of about 5-fold) for the different ligands. This is not surprising because partial saturation of the Gal/GalNAc receptor on the surface of rabbit hepatocytes with a macromolecular ligand (ASOR) affects the binding ofgalactose-terminated ligands to the remaining accessible receptors in a specific manner (25).…”

“…Ligands were radioiodinated by a modification of the chloramine-T method (24). Direct binding assays were carried out as previously outlined, and nonspecific binding was routinely assessed by using a nondiluting EDTA release protocol (25). The data were analyzed by using a modified version of the nonlinear multiparameter regression program LIGAND (26) as described (25).…”

“…Direct binding assays were carried out as previously outlined, and nonspecific binding was routinely assessed by using a nondiluting EDTA release protocol (25). The data were analyzed by using a modified version of the nonlinear multiparameter regression program LIGAND (26) as described (25). Nonspecific binding was calculated based on the LIGAND-determined value of the N parameter, which is the limiting [bound]/[free] ligand ratio.…”