Preparation and characterization of chemical affinity probes for the hepatic galactosyl receptor
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), Vol. 45:6; Conference: 76. annual meeting of the Federation of American Society for Experimental Biology, Washington, DC, USA, 8 Jun 1986
- Country of Publication:
- United States
- Language:
- English
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Related Subjects
GALACTOSE
RECEPTORS
SACCHARIDES
BIOCHEMICAL REACTION KINETICS
CROSS-LINKING
AUTORADIOGRAPHY
COVALENCE
ELECTROPHORESIS
IODINE 125
LIGANDS
ALDEHYDES
BETA DECAY RADIOISOTOPES
CARBOHYDRATES
CHEMICAL REACTIONS
DAYS LIVING RADIOISOTOPES
ELECTRON CAPTURE RADIOISOTOPES
HEXOSES
INTERMEDIATE MASS NUCLEI
IODINE ISOTOPES
ISOTOPES
KINETICS
MEMBRANE PROTEINS
MONOSACCHARIDES
NUCLEI
ODD-EVEN NUCLEI
ORGANIC COMPOUNDS
POLYMERIZATION
PROTEINS
RADIOISOTOPES
REACTION KINETICS
550201* - Biochemistry- Tracer Techniques