Characterization of an intracellular hyaluronic acid binding site in isolated rat hepatocytes

• Published in: Biochemistry (Easton) 1990-11, Vol.29 (45), p.10425-10432
• Main Authors: Frost, Stephen J, Raja, Rampyari H, Weigel, Paul H
• Format: Article
• Language: English
• Subjects: 550201 - Biochemistry- Tracer Techniques; AMINES; Analytical, structural and metabolic biochemistry; ANIMAL CELLS; ANIMALS; BASIC BIOLOGICAL SCIENCES; BETA DECAY RADIOISOTOPES; Binding and carrier proteins; Binding Sites; BIOCHEMICAL REACTION KINETICS; Biological and medical sciences; CARBOHYDRATES; Carrier Proteins - metabolism; CELL CONSTITUENTS; Cell Membrane Permeability; CELL MEMBRANES; Cells, Cultured; CHEMICAL COMPOSITION; DAYS LIVING RADIOISOTOPES; Digitonin - pharmacology; Egtazic Acid - pharmacology; ELECTRON CAPTURE RADIOISOTOPES; Endocytosis; Endothelium - drug effects; Endothelium - metabolism; Fundamental and applied biological sciences. Psychology; Glycosaminoglycans - metabolism; Hyaluronan Receptors; HYALURONIC ACID; Hyaluronic Acid - metabolism; INTERMEDIATE MASS NUCLEI; IODINE 125; IODINE ISOTOPES; Iodine Radioisotopes; ISOTOPE APPLICATIONS; ISOTOPES; KINETICS; Liver - drug effects; Liver - metabolism; LIVER CELLS; Male; MAMMALS; MEMBRANE PROTEINS; MEMBRANES; MUCOPOLYSACCHARIDES; NUCLEI; ODD-EVEN NUCLEI; ORGANIC COMPOUNDS; PERMEABILITY; POLYSACCHARIDES; PROTEINS; RADIOISOTOPES; RATS; Rats, Inbred Strains; REACTION KINETICS; RECEPTORS; RODENTS; SACCHARIDES; SOMATIC CELLS; TRACER TECHNIQUES; VERTEBRATES
• ISSN: 0006-2960; 1520-4995
• DOI: 10.1021/bi00497a019

125I-HA, prepared by chemical modification at the reducing sugar, specifically binds to rat hepatocytes in suspension or culture. Intact hepatocytes have relatively few surface 125I-HA binding sites and show low specific binding. However, permeabilization of hepatocytes with the nonionic detergent digitonin results in increased specific 125I-HA binding (45-65%) and a very large increase in the number of specific 125I-HA binding sites. Scatchard analysis of equilibrium 125I-HA binding to permeabilized hepatocytes in suspension at 4 degrees C indicates a Kd = 1.8 x 10(-7) M and 1.3 x 10(6) molecules of HA (Mr approximately 30,000) bound per cell at saturation. Hepatocytes in primary culture for 24 h show the same affinity but the total number of HA molecules bound per cell at saturation decreases to approximately 6.2 x 10(5). Increasing the ionic strength above physiologic concentrations decreases 125I-HA binding to permeable cells, whereas decreasing the ionic strength above causes an approximately 4-fold increase. The divalent cation chelator EGTA does not prevent binding nor does it release 125I-HA bound in the presence of 2 mM CaCl2, although higher divalent cation concentrations stimulate 125I-HA binding. Ten millimolar CaCl2 or MnCl2 increases HA binding 3-6-fold compared to EGTA-treated cells. Ten millimolar MgCl2, SrCl2, or BaCl2 increased HA binding by 2-fold. The specific binding of 125I-HA to digitonin-treated hepatocytes at 4 degrees C increased greater than 10-fold at pH 5.0 as compared to pH 7.