Chi-Rosso G, Toole B P
J Cell Biochem. 1987 Mar;33(3):173-83. doi: 10.1002/jcb.240330304.
Hyaluronate-binding protein (HABP) has been extracted in detergent from the membranes of simian virus 40-transformed 3T3 (SV-3T3) cells (Underhill et al, J Biol Chem 258:8086-8091, 1983). When SV-3T3 cells were treated with trypsin prior to isolation and dissolution of the membranes, no hyaluronate-binding activity could be detected. This indicates that all of the detectable HABP of SV-3T3 cells is located on the external surface of the plasma membrane rather than on internal membranes, which would be inaccessible to the trypsin. The detergent-extracted HABP from SV-3T3 membranes was reconstituted into the membrane of lipid vesicles, which were formed by addition of exogenous phosphatidylcholine and cholic acid to the extracts followed by removal of detergent by dialysis against 0.02 M Tris pH 8.0 in the presence of protease inhibitors. Reconstitution was assessed by sedimentation in a discontinuous sucrose gradient and by gel filtration on Sepharose 4B in the presence and absence of detergent. The characteristics of binding of hyaluronate to the reconstituted HABP were then compared with those studied previously for the original membrane-bound HABP and the detergent-extracted HABP (Underhill et al, J Biol Chem 258:8086-8091, 1983). It was observed previously that binding of hyaluronate to HABP in the cell membranes was of higher affinity and specificity than to HABP in the detergent extracts of these membranes. It was found here that reconstitution of the extracted HABP into the membranes of lipid vesicles led to restoration of affinity of binding to the level observed in the original cell membranes. However, whereas chondroitin sulfate does not compete significantly for binding of hyaluronate to cell membrane-bound HABP, partial competition was observed for the reconstituted HABP as well as for detergent-extracted HABP. Thus, it is concluded that the high affinity of binding of hyaluronate to the plasma membrane of SV-3T3 cells is in part dependent on insertion of the HABP in the membrane, but that other interactions, not duplicated in our reconstitution experiments, must be necessary for the specificity of the HABP.
透明质酸结合蛋白（HABP）已用去污剂从猿猴病毒40转化的3T3（SV - 3T3）细胞的膜中提取出来（安德希尔等人，《生物化学杂志》258:8086 - 8091，1983年）。当在分离和溶解膜之前用胰蛋白酶处理SV - 3T3细胞时，无法检测到透明质酸结合活性。这表明SV - 3T3细胞中所有可检测到的HABP都位于质膜的外表面，而不是位于内部膜上，因为内部膜对胰蛋白酶是不可接近的。从SV - 3T3膜中用去污剂提取的HABP被重新组装到脂质体膜中，脂质体是通过向提取物中添加外源性磷脂酰胆碱和胆酸，然后在蛋白酶抑制剂存在的情况下用0.02 M Tris pH 8.0进行透析去除去污剂而形成的。通过在不连续蔗糖梯度中沉降以及在有无去污剂的情况下在琼脂糖4B上进行凝胶过滤来评估重新组装情况。然后将透明质酸与重新组装的HABP的结合特性与先前针对原始膜结合HABP和去污剂提取的HABP研究的特性进行比较（安德希尔等人，《生物化学杂志》258:8086 - 8091，1983年）。先前观察到，透明质酸与细胞膜中HABP的结合比与这些膜的去污剂提取物中的HABP具有更高的亲和力和特异性。在此发现，将提取的HABP重新组装到脂质体膜中导致结合亲和力恢复到原始细胞膜中观察到的水平。然而，虽然硫酸软骨素对透明质酸与细胞膜结合的HABP的结合没有显著竞争，但对重新组装的HABP以及去污剂提取的HABP都观察到了部分竞争。因此，可以得出结论，透明质酸与SV - 3T3细胞质膜的高亲和力结合部分取决于HABP在膜中的插入，但对于HABP的特异性来说，我们的重新组装实验中未重复的其他相互作用必定是必要的。
