Preparation and characterization of chemical affinity probes for the hepatic galactosyl receptor

Journal Article · Thu May 01 04:00:00 EDT 1986 · Fed. Proc., Fed. Am. Soc. Exp. Biol.; (United States)

OSTI ID:6937164

Herzig, M C; Weigel, P H

The authors have developed a protocol to covalently attach asialo-orosomucoid (ASOR) to the Galactosyl receptor. /sup 125/I-ASOR was modified to an NHS ester by reaction with either disuccinimidyl suberate to give NHS-/sup 125/I-ASOR or dithiobis(succinimidylpropionate) to give NHS-S-S/sup 125/I-ASOR. The specificity of binding of the purified derivatives to isolated rat hepatocytes at 4/sup 0/C, assessed in the presence of ASOR, was >90%. Derivatized ASOR was shown to be covalently cross-linked by the inability of EGTA, to release radioactivity. Optimum cross-linking of the specifically bound derivative was greater than or equal to40%. The specificity of cross-linking to receptor with both cross-linkers was approx.80% as determined by immunoprecipitation of EGTA-treated cells with a goat anti-receptor antisera. SDS-PAGE and autoradiography of extracts from cells cross-linked with either derivative revealed bands of M/sub r/ approx. 108-110 kDa, and 82 kDa under both reducing and nonreducing conditions. Thus, receptor and ASOR could be cross-linked with both high efficiency and high specificity. In order to assess cellular internalization of cross-linked receptor-ligand complexes, cells were treated with dithiothreitol or trypsin to remove, respectively, only the surface bound NHS-S-S-/sup 125/I-ASOR or NHS-/sup 125/I-ASOR. The kinetics of internalization for both derivatized or underivatized ASOR were identical. These results directly show for the first time that both surface receptor and ligand are internalized.

• Research Organization: Univ. of Texas Medical Branch, Galveston
• OSTI ID: 6937164
• Report Number(s): CONF-8606151-
• Journal Information: Fed. Proc., Fed. Am. Soc. Exp. Biol.; (United States), Journal Name: Fed. Proc., Fed. Am. Soc. Exp. Biol.; (United States) Vol. 45:6; ISSN FEPRA
• Country of Publication: United States
• Language: English

Related Subjects

550201* -- Biochemistry-- Tracer Techniques
59 BASIC BIOLOGICAL SCIENCES
ALDEHYDES
AUTORADIOGRAPHY
BETA DECAY RADIOISOTOPES
BIOCHEMICAL REACTION KINETICS
CARBOHYDRATES
CHEMICAL REACTIONS
COVALENCE
CROSS-LINKING
DAYS LIVING RADIOISOTOPES
ELECTRON CAPTURE RADIOISOTOPES
ELECTROPHORESIS
GALACTOSE
HEXOSES
INTERMEDIATE MASS NUCLEI
IODINE 125
IODINE ISOTOPES
ISOTOPES
KINETICS
LIGANDS
MEMBRANE PROTEINS
MONOSACCHARIDES
NUCLEI
ODD-EVEN NUCLEI
ORGANIC COMPOUNDS
POLYMERIZATION
PROTEINS
RADIOISOTOPES
REACTION KINETICS
RECEPTORS
SACCHARIDES