Effects of detergent solubilization on the hyaluronate-binding protein from membranes of simian virus 40-transformed 3T3 cells.
Effects of detergent solubilization on the hyaluronate-binding protein from membranes of simian virus 40-transformed 3T3 cells.
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发表时间：
1983-07
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通讯作者：
C. Underhill;G. Chi‐Rosso;B. Toole
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作者：
C. Underhill;G. Chi‐Rosso;B. Toole
猴病毒40转化的3 T3（SV-3 T3）细胞已显示在其表面上具有透明质酸的结合位点，其介导这些细胞的二价阳离子非依赖性聚集。为了进一步表征这些结合位点，从SV-3 T3细胞制备膜并用去污剂脱氧胆酸钠溶解。通过加入[3 H]透明质酸盐，然后通过（NH 4）2SO 4沉淀分离游离和结合的配体，测量洗涤剂溶液中存在的结合活性。使用该测定，将可溶性透明质酸结合蛋白与原位膜相关蛋白进行比较。在这两种情况下，发现结合是饱和的，与蛋白质含量呈线性关系，受到未标记透明质酸盐的竞争性抑制，并依赖于透明质酸盐的至少6个糖残基进行识别。然而，发现溶解的结合蛋白与膜结合蛋白在以下特征上不同：a）与透明质酸的相互作用的亲和力降低（Kd增大），而饱和结合量（Bmax）增大; B）在用未标记的透明质酸盐制剂的竞争实验中，透明质酸盐的MWv对其抑制效力的影响大大降低;和c）硫酸软骨素和硫酸皮肤素在更大程度上抑制结合。所有这些差异都可以通过假设去污剂溶解将透明质酸盐结合位点相互作用的性质从多价（即一个透明质酸盐分子连接到几个位点）改变为单价来解释。
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.