Fuchs H E, Michalopoulos G K, Pizzo S V
J Cell Biochem. 1984;25(4):231-43. doi: 10.1002/jcb.240250405.
In vivo clearance studies have indicated that the clearance of proteinase complexes of the homologous serine proteinase inhibitors alpha 1-proteinase inhibitor and antithrombin III occurs via a specific and saturable pathway located on hepatocytes. In vitro hepatocyte-uptake studies with antithrombin III-proteinase complexes confirmed the hepatocyte uptake and degradation of these complexes, and demonstrated the formation of a disulfide interchange product between the ligand and a cellular protein. We now report the results of in vitro hepatocyte uptake studies with alpha 1-proteinase inhibitor-trypsin complexes. Trypsin complexes of alpha 1-proteinase inhibitor were prepared and purified to homogeneity. Uptake of these complexes by hepatocytes was time and concentration-dependent. Competition experiments with alpha 1-proteinase inhibitor, alpha 1-proteinase inhibitor-trypsin, and antithrombin III-thrombin indicated that the proteinase complexes of these two inhibitors are recognized by the same uptake mechanism, whereas the native inhibitor is not. Uptake studies were performed at 37 degrees C with 125I-alpha 1-proteinase inhibitor-trypsin and analyzed by sodium dodecyl sulfate-gel electrophoresis in conjunction with autoradiography. These studies demonstrated time-dependent uptake and degradation of the ligand to low molecular weight peptides. In addition, there was a time-dependent accumulation of a high molecular weight complex of ligand and a cellular protein. This complex disappeared when gels were performed under reducing conditions. The sole cysteine residue in alpha 1-proteinase inhibitor was reduced and alkylated with iodoacetamide. Trypsin complexes of the modified inhibitor were prepared and purified to homogeneity. Uptake and degradation studies demonstrated no differences in the results obtained with this modified complex as compared to unmodified alpha 1-proteinase inhibitor-trypsin complex. In addition, the high molecular weight disulfide interchange product was still present on sodium dodecyl sulfate-polyacrylamide gel electrophoresis of solubilized cells. Clearance and clearance competition studies with alpha 1-proteinase inhibitor-trypsin, alkylated alpha 1-proteinase inhibitor-trypsin, antithrombin III-thrombin, and anti-thrombin III-factor IXa further demonstrated the shared hepatocyte uptake mechanism for all these complexes.
体内清除研究表明，同源丝氨酸蛋白酶抑制剂α1-蛋白酶抑制剂和抗凝血酶III的蛋白酶复合物通过位于肝细胞上的特定且可饱和的途径进行清除。用抗凝血酶III-蛋白酶复合物进行的体外肝细胞摄取研究证实了这些复合物的肝细胞摄取和降解，并证明了配体与细胞蛋白之间形成了二硫键交换产物。我们现在报告用α1-蛋白酶抑制剂-胰蛋白酶复合物进行的体外肝细胞摄取研究结果。制备并纯化α1-蛋白酶抑制剂的胰蛋白酶复合物至同质。肝细胞对这些复合物的摄取具有时间和浓度依赖性。用α1-蛋白酶抑制剂、α1-蛋白酶抑制剂-胰蛋白酶和抗凝血酶III-凝血酶进行的竞争实验表明，这两种抑制剂的蛋白酶复合物通过相同的摄取机制被识别，而天然抑制剂则不然。在37℃下用125I-α1-蛋白酶抑制剂-胰蛋白酶进行摄取研究，并通过十二烷基硫酸钠-凝胶电泳结合放射自显影进行分析。这些研究表明配体随时间摄取并降解为低分子量肽。此外，配体与细胞蛋白的高分子量复合物有时间依赖性的积累。当在还原条件下进行凝胶电泳时，该复合物消失。α1-蛋白酶抑制剂中的唯一半胱氨酸残基用碘乙酰胺还原并烷基化。制备并纯化修饰抑制剂的胰蛋白酶复合物至同质。摄取和降解研究表明，与未修饰的α1-蛋白酶抑制剂-胰蛋白酶复合物相比，用这种修饰复合物获得的结果没有差异。此外，在溶解细胞的十二烷基硫酸钠-聚丙烯酰胺凝胶电泳上仍存在高分子量二硫键交换产物。用α1-蛋白酶抑制剂-胰蛋白酶、烷基化α1-蛋白酶抑制剂-胰蛋白酶、抗凝血酶III-凝血酶和抗凝血酶III-因子IXa进行的清除和清除竞争研究进一步证明了所有这些复合物共有的肝细胞摄取机制。
