Characterization of an intracellular hyaluronic acid binding site in isolated rat hepatocytes.
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.(ABSTRACT TRUNCATED AT 250 WORDS)
Frost
S J
SJ
University of Texas Medical Branch, Department of Human Biological Chemistry and Genetics, Galveston 77550.
Raja
R H
RH
Weigel
P H
PH
D000818:Animals; D001665:Binding Sites; D002352:Carrier Proteins / Q000378:metabolism*; D002463:Cell Membrane Permeability; D002478:Cells, Cultured; D004072:Digitonin / Q000494:pharmacology; D004533:Egtazic Acid / Q000494:pharmacology; D004705:Endocytosis; D004727:Endothelium / Q000187:drug effects / Q000378:metabolism; D006025:Glycosaminoglycans / Q000378:metabolism; D018960:Hyaluronan Receptors; D006820:Hyaluronic Acid / Q000378:metabolism; D007457:Iodine Radioisotopes; D007700:Kinetics; D008099:Liver / Q000187:drug effects / Q000378:metabolism*; D008297:Male; D051381:Rats; D011919:Rats, Inbred Strains
D002352:Carrier Proteins; D006025:Glycosaminoglycans; D018960:Hyaluronan Receptors; D007457:Iodine Radioisotopes; D004533:Egtazic Acid; D006820:Hyaluronic Acid; D004072:Digitonin