Specific intracellular hyaluronic acid binding to isolated rat hepatocytes is membrane-associated.
Intact isolated rat hepatocytes show a small amount of specific 125I-labeled hyaluronic acid (HA) binding. However, in the presence of digitonin, a very large increase in the specific binding of 125I-HA is observed. Chondroitin sulfate, heparin and dextran sulfate were as effective as unlabeled HA in competing for 125I-HA binding to permeabilized hepatocytes, indicating that the binding sites may have a general specificity for glycosaminoglycans. After rat hepatocytes had been homogenized in a hypotonic buffer, more than 98% of the 125I-HA binding activity could be pelleted by centrifugation at 100,000 x g for 1 h. Mild alkaline treatment of hepatocyte membranes did not release 125I-HA binding activity, suggesting that the HA binding site is an integral membrane molecule. Furthermore, trypsin treatment of deoxycholate-extracted membranes destroyed the binding activity, as assessed by a dot-blot assay. This suggests that a protein component in the membrane is necessary for 125I-HA binding activity. Rat fibrinogen could be a possible candidate for the HA binding activity because HA binds specifically to human fibrinogen (LeBoeuf et al. (1986) J. Biol. Chem. 261, 12 586). Also, fibrinogen can be found in a quasi-crystalline form in rat hepatocytes and could be pelleted with the membranes. Rat fibrinogen was not responsible for the 125I-HA binding activity, since (1) purified rat fibrinogen did not bind to 125I-HA, and (2) immunoprecipitation of rat fibrinogen from hepatocyte extracts did not decrease the 125I-HA binding of these extracts. We conclude that the internal HA binding sites are membrane- or cytoskeleton-associated proteins and are neither cytosolic proteins nor fibrinogen.
Frost
S J
SJ
Department of Human Biological Chemistry and Genetics, University of Texas Medical Branch, Galveston 77550.
McGary
C T
CT
Raja
R H
RH
Weigel
P H
PH
D000818:Animals; D015153:Blotting, Western; D002254:Carbonates / Q000494:pharmacology; D002463:Cell Membrane Permeability; D004072:Digitonin / Q000494:pharmacology; D005340:Fibrinogen / Q000378:metabolism; D006820:Hyaluronic Acid / Q000378:metabolism*; D008099:Liver / Q000378:metabolism*; D008297:Male; D008566:Membranes / Q000378:metabolism; D051381:Rats; D011919:Rats, Inbred Strains; D014357:Trypsin / Q000378:metabolism
D002254:Carbonates; C005686:sodium carbonate; D005340:Fibrinogen; D006820:Hyaluronic Acid; D014357:Trypsin; D004072:Digitonin