Effects of detergent solubilization on the hyaluronate-binding protein from membranes of simian virus 40-transformed 3T3 cells.
Simian virus 40-transformed 3T3 (SV-3T3) cells have been shown to possess on their surfaces binding sites for hyaluronate which mediate the divalent cation independent aggregation of these cells. To further characterize these binding sites, membranes were prepared from SV-3T3 cells and solubilized with the detergent, sodium deoxycholate. The binding activity present in the detergent solution was measured by the addition of [3H]hyaluronate followed by separation of free and bound ligand by (NH4)2SO4 precipitation. Using this assay, the soluble hyaluronate-binding protein was compared with the membrane-associated protein in situ. In both cases, binding was found to be saturable, linear with protein content, competitively inhibited by unlabeled hyaluronate and dependent on a minimum of 6 sugar residues of hyaluronate for recognition. However, the solubilized binding protein was found to differ from the membrane-associated protein in the following characteristics: a) the affinity of the interaction with hyaluronate was reduced (the Kd was higher), while the amount of ligand bound at saturation (Bmax) was increased; b) in competition experiments with unlabeled preparations of hyaluronate, the effect that the MWv of the hyaluronate had on its inhibitory potency was greatly reduced; and c) the binding was inhibited to a greater extent by chondroitin sulfate and dermatan sulfate. All of these differences can be accounted for by assuming that the detergent solubilization changes the nature of the hyaluronate-binding site interaction from one that is multivalent (i.e. one molecule of hyaluronate is attached to several sites) to one that is monovalent.
D000818:Animals; D002352:Carrier Proteins / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism; D002462:Cell Membrane / Q000378:metabolism; D002471:Cell Transformation, Neoplastic; D002478:Cells, Cultured; D003840:Deoxycholic Acid; D018960:Hyaluronan Receptors; D006820:Hyaluronic Acid / Q000235:genetics* / Q000378:metabolism; D007700:Kinetics; D051379:Mice; D009844:Oligosaccharides; D013539:Simian virus 40 / Q000235:genetics*; D012995:Solubility
D002352:Carrier Proteins; D018960:Hyaluronan Receptors; D009844:Oligosaccharides; D003840:Deoxycholic Acid; D006820:Hyaluronic Acid