Detergent solubilization of the endocytic Ca(2+)-independent hyaluronan receptor from rat liver endothelial cells and separation from a Ca(2+)-dependent hyaluronan-binding activity.
Rat liver sinusoidal endothelial cells (LECs) mediate the removal of hyaluronan (HA) from the circulation via a specific Ca(2+)-independent endocytic receptor. To characterize the receptor biochemically, detergent-soluble extracts were prepared from crude LEC membranes. Using a dot blot assay to quantitate 125I-HA binding activity in CHAPS-solubilized membranes, we detected not only specific Ca(2+)-independent but also specific Ca(2+)-dependent HA-binding activity. Both HA-binding activities behave as integral membrane-associated proteins; they are not released from LEC membranes by treatment at pH 11, and they require detergent for extraction. The Ca(2+)-independent HA receptor was inactivated by treatment at 56 degrees C for 30 min or with 200 mM DTT at 4 degrees C for 30 min, whereas the Ca(2+)-dependent activity actually increased by 75% after treatment at 56 degrees C and only 20% of the Ca(2+)-dependent activity was lost after DTT treatment. A two-cycle membrane extraction protocol using CHAPS partially separated the two HA-binding activities. Eight millimolar KCl and 0.5% CHAPS extracted approximately 50% of the Ca(2+)-independent HA receptor, but only 4-11% of the Ca(2+)-dependent activity. When the KCl and CHAPS concentrations were increased to 2.0 M and 1.5%, respectively, the remaining HA receptor, as well as 89-96% of the Ca(2+)-dependent activity, was then extracted. The Ca(2+)-independent and Ca(2+)-dependent activities could also be further separated using Sephacryl S-400 gel filtration chromatography.(ABSTRACT TRUNCATED AT 250 WORDS)
Department of Human Biological Chemistry and Genetics, University of Texas Medical Branch, Galveston 77550.
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Yannariello-Brown
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Weigel
D000818:Animals; D002118:Calcium / Q000494:pharmacology*; D002254:Carbonates / Q000494:pharmacology; D002352:Carrier Proteins / Q000032:analysis*; D002793:Cholic Acids; D002850:Chromatography, Gel; D004705:Endocytosis; D004727:Endothelium / Q000737:chemistry*; D006801:Humans; D018960:Hyaluronan Receptors; D006820:Hyaluronic Acid / Q000378:metabolism*; D006863:Hydrogen-Ion Concentration; D008099:Liver / Q000737:chemistry*; D008297:Male; D011485:Protein Binding; D051381:Rats; D011919:Rats, Inbred Strains; D011956:Receptors, Cell Surface / Q000032:analysis* / Q000737:chemistry / Q000187:drug effects; D012995:Solubility; D013696:Temperature
D002254:Carbonates; D002352:Carrier Proteins; D002793:Cholic Acids; D018960:Hyaluronan Receptors; D011956:Receptors, Cell Surface; C005686:sodium carbonate; D006820:Hyaluronic Acid; C028213:3-((3-cholamidopropyl)dimethylammonium)-1-propanesulfonate; D002118:Calcium