Frost S J, McGary C T, Raja R H, Weigel P H
Department of Human Biological Chemistry and Genetics, University of Texas Medical Branch, Galveston 77550.
Biochim Biophys Acta. 1988 Dec 8;946(1):66-74. doi: 10.1016/0005-2736(88)90458-0.
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.
完整分离的大鼠肝细胞显示出少量特异性的125I标记透明质酸（HA）结合。然而，在存在洋地黄皂苷的情况下，观察到125I-HA的特异性结合大幅增加。硫酸软骨素、肝素和硫酸葡聚糖在竞争125I-HA与通透化肝细胞的结合方面与未标记的HA一样有效，这表明结合位点可能对糖胺聚糖具有普遍特异性。大鼠肝细胞在低渗缓冲液中匀浆后，超过98%的125I-HA结合活性可通过在100,000×g下离心1小时沉淀下来。对肝细胞膜进行温和碱性处理不会释放125I-HA结合活性，这表明HA结合位点是一种整合膜分子。此外，通过斑点印迹分析评估，用胰蛋白酶处理脱氧胆酸盐提取的膜会破坏结合活性。这表明膜中的蛋白质成分对于125I-HA结合活性是必需的。大鼠纤维蛋白原可能是HA结合活性的一个候选者，因为HA特异性结合人纤维蛋白原（勒伯夫等人（1986年）《生物化学杂志》261卷，12586页）。此外，纤维蛋白原可以以准晶体形式存在于大鼠肝细胞中，并且可以与膜一起沉淀下来。大鼠纤维蛋白原不负责125I-HA结合活性，因为（1）纯化的大鼠纤维蛋白原不与125I-HA结合，并且（2）从肝细胞提取物中免疫沉淀大鼠纤维蛋白原不会降低这些提取物的125I-HA结合。我们得出结论，内部HA结合位点是与膜或细胞骨架相关的蛋白质，既不是胞质蛋白也不是纤维蛋白原。
