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 00:00:00 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), Vol. 28:2, Issue 2; ISSN 0006-2960
• Country of Publication: United States
• Language: English

Related Subjects

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