Andus T, Gross V, Tran-Thi T A, Schreiber G, Nagashima M, Heinrich P C
Eur J Biochem. 1983 Jul 1;133(3):561-71. doi: 10.1111/j.1432-1033.1983.tb07500.x.
The biosynthesis and secretion of alpha 2-macroglobulin, transferrin, alpha 1-acid glycoprotein and alpha 1-proteinase inhibitor were studied in rat hepatocyte primary cultures. After labeling with [35S]methionine, two forms, which can be separated electrophoretically differing by molecular weight, were found for each of the four glycoproteins. The following molecular weights were estimated for the intracellular precursors and the secreted forms: alpha 2-macroglobulin, 176 000 and 182 000; transferrin, 84 000 and 86 000; alpha 1-acid glycoprotein, 39 000 and 43 000-60 000; alpha 1-proteinase inhibitor, 49 000 and 54 000. Carbohydrate moieties could be removed from intracellular forms by treatment with endoglucosaminidase H indicating that their oligosaccharide chains were of the high-mannose type. The extracellular forms were sensitive to sialidase. They incorporated [3H]galactose and [3H]fucose showing that their oligosaccharide chains were of the complex type. Pulse-chase experiments revealed a precursor-product relationship for the high-mannose and the complex type glycoproteins. In the hepatocyte medium newly synthesized albumin was detected after 30 min and newly synthesized glycoproteins after 60 min. Unglycosylated alpha 2-macroglobulin (162 000), transferrin (79 000), alpha 1-acid glycoprotein (23 000), and alpha 1-proteinase inhibitor (41 000) were found in the cells as well as in the medium, when the transfer of oligosaccharide chains onto the polypeptide chains was blocked by tunicamycin. Tunicamycin led to a marked reduction of the secretion of alpha 2-macroglobulin, alpha 1-acid glycoprotein and alpha 1-proteinase inhibitor, whereas the secretion of transferrin was less affected.
在大鼠肝细胞原代培养物中研究了α2-巨球蛋白、转铁蛋白、α1-酸性糖蛋白和α1-蛋白酶抑制剂的生物合成及分泌。用[35S]甲硫氨酸标记后，发现这四种糖蛋白中的每一种都有两种可通过电泳分离的形式，它们的分子量不同。对细胞内前体和分泌形式估计的分子量如下：α2-巨球蛋白，176000和182000；转铁蛋白，84000和86000；α1-酸性糖蛋白，39000和43000 - 60000；α1-蛋白酶抑制剂，49000和54000。用内切葡糖胺酶H处理可从细胞内形式中去除碳水化合物部分，这表明它们的寡糖链是高甘露糖型的。细胞外形式对唾液酸酶敏感。它们掺入[3H]半乳糖和[3H]岩藻糖，表明它们的寡糖链是复合型的。脉冲追踪实验揭示了高甘露糖型和复合型糖蛋白的前体-产物关系。在肝细胞培养基中，30分钟后检测到新合成的白蛋白，60分钟后检测到新合成的糖蛋白。当寡糖链向多肽链的转移被衣霉素阻断时，在细胞和培养基中都发现了未糖基化的α2-巨球蛋白（162000）、转铁蛋白（79000）、α1-酸性糖蛋白（23000）和α1-蛋白酶抑制剂（41000）。衣霉素导致α2-巨球蛋白、α1-酸性糖蛋白和α1-蛋白酶抑制剂的分泌显著减少，而转铁蛋白的分泌受影响较小。
