Different Modes of Ligand Binding to the Hepatic Galactose/N-Acetylgalactosamine Lectin on the Surface of Rabbit Hepatocytes

Mark R. Hardy, R. Reid Townsend, S. M. Parkhurst, Yuan Chuan Lee

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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 161000 sites/cell with a dissociation constant of 0.44 nM and 292000 sites/cell with a Kd of 9.7 nM. Asialotriantennary glycopeptide purified from human α-1 protease inhibitor and modified with tyrosine at the N-terminus to permit radioiodination (TRI) [Lee, Y. C., Townsend, R. R., Hardy, M. R., Lonngren, J., Arnarp, J., Haraldsson, M., & Lonn, 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 982000 sites/cell of Kd = 25.3 nM. A synthetic ligand, a,/?-diaspartamide of tris[(β-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 817000 sites/cell of KA = 0.63 nM and 1.23 x 106 sites/cell of KA = 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. The nature of the additional TRI or di-tris-lac binding sites was investigated by measuring the binding of TRI or di-tris-lac to rabbit hepatocytes that had their high-affinity ASOR sites occupied by unlabeled ASOR (ASOR-loaded cells). ASOR-loaded cells were found to bind ASOR or TRI to a single class of remaining low-affinity sites (Kd = 38.0 nM for ASOR and KA- 10.2 nM for TRI), with an approximate stoichiometry equivalent to three TRI sites per ASOR site (171000 ASOR sites/cell vs. 495 000 TRI sites/cell). Di-tris-lac, however, was still observed to bind with high affinity (Kd = 1.2 nM) to 87 900 sites/cell, as well as to 429000 low-affinity (ATd = 50.0 nM) sites/cell. The high-affinity di-tris-lac sites appear to be unavailable for high-affinity TRI or ASOR binding. We concluded that the sites that accommodate one galactose residue are clustered on the cell surface, permitting ASOR to bind to or eclipse about nine galactose-combining sites. However, ASOR does not delineate all of the high-affinity binding sites on the cell surface, as defined by the small ligands TRI and di-tris-lac. We explained these observations by considering that ligands bind to a lattice of galactose-combining sites on the cell surface and that both the apparent number and affinity of binding sites are a function of the ligand utilized.

Original languageEnglish
Pages (from-to)22-28
Number of pages7
JournalBiochemistry
Volume24
Issue number1
DOIs
StatePublished - Jan 1 1985

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