Synthesis and characterization of N-hydroxysuccinimide ester chemical affinity derivatives of asialoorosomucoid that covalently cross-link to galactosyl receptors on isolated rat hepatocytes

Journal Article · Tue Jan 24 04:00:00 EST 1989 · Biochemistry; (USA)

DOI:https://doi.org/10.1021/bi00428a028· OSTI ID:5482307

Herzig, M C.S.; Weigel, P H ^[1]

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1. Univ. of Texas Medical Branch, Galveston (USA)

The authors have developed chemical affinity reagents for the hepatic galactosyl receptor. Asialoorosomucoid (ASOR) was derivatized with five homobifunctional N-hydroxysuccinimide (NHS) ester cross-linkers. NHS/ASOR derivatives were synthesized, purified, and applied within 10 min to isolated rat hepatocytes at 4{degree}C. Specific binding of these {sup 125}I-labeled derivatives was {approximately}90% in the presence of either EGTA or excess ASOR. Specific cross-linking assessed by the resistance of specifically bound NHS/{sup 125}I-ASOR to release by EGTA, was 50-75% of the specifically bound ligand. The extent of specific cross-linking correlated with the average number of NHS groups per ASOR and was controlled by varying the molar ratio of cross-linker to ASOR during the synthesis. After being cross-linked with any of the NHS/{sup 125}I-ASOR derivatives, cells were washed with EGTA, solubilized in Triton X-100, and analyzed by SDA-PAGE and autoradiography. They conclude that all three receptor subunits can cross-link to ligand. They propose a model in which the native receptor is a heterohexamer composed of four subunits of RHL 1 and two subunits of RHL 2 and/or RHL 3.

• OSTI ID: 5482307
• Journal Information: Biochemistry; (USA), Journal Name: Biochemistry; (USA) Journal Issue: 2 Vol. 28:2; ISSN 0006-2960; ISSN BICHA
• Country of Publication: United States
• Language: English

Related Subjects

550201* -- Biochemistry-- Tracer Techniques
59 BASIC BIOLOGICAL SCIENCES
ALKALI METAL COMPOUNDS
ANIMAL CELLS
AUTORADIOGRAPHY
BETA DECAY RADIOISOTOPES
BIOCHEMICAL REACTION KINETICS
BIOSYNTHESIS
CARBOHYDRATES
CARBOXYLIC ACIDS
CHEMICAL REACTIONS
CROSS-LINKING
DAYS LIVING RADIOISOTOPES
DERIVATIZATION
DICARBOXYLIC ACIDS
ELECTRON CAPTURE RADIOISOTOPES
ELECTROPHORESIS
ESTERS
GLUCOPROTEINS
GLYCOPROTEINS
HALIDES
HALOGEN COMPOUNDS
INORGANIC PHOSPHORS
INTERMEDIATE MASS NUCLEI
IODIDES
IODINE 125
IODINE COMPOUNDS
IODINE ISOTOPES
ISOTOPES
KINETICS
LIGANDS
LIVER CELLS
MEMBRANE PROTEINS
MOLECULAR STRUCTURE
NUCLEI
ODD-EVEN NUCLEI
ORGANIC ACIDS
ORGANIC COMPOUNDS
PH VALUE
PHOSPHORS
POLYMERIZATION
PROTEINS
RADIOISOTOPES
REACTION KINETICS
RECEPTORS
SACCHARIDES
SODIUM COMPOUNDS
SODIUM IODIDES
SOMATIC CELLS
SUCCINIC ACID
SYNTHESIS