Cooper A R, Kurkinen M, Taylor A, Hogan B L
Eur J Biochem. 1981 Sep;119(1):189-97. doi: 10.1111/j.1432-1033.1981.tb05593.x.
The biosynthesis and processing of the polypeptides A (Mr = 450 x 10(3)), B1 (Mr = 240 x 10(3)), B2 (Mr = 230 x 10(3)) and C (Mr = 150 x 10(3)) of the extracellular matrix protein, laminin, were studied in murine parietal endoderm cells labelled with [35S]methionine. Various lines of evidence suggest that the A chains are not precursors to the smaller B chains. Firstly, the pulse-chase experiments, radioactivity in cytoplasmic A and (B1 + B2) chains declines with the same half-life of about 70 min. Secondly, peptide maps generated by digestion of A and B (B1 + B2) chains with Staphylococcus aureus V8 protease are different. Finally, rabbit antibodies to isolated, denatured (B1 + B2) chains do not cross-react with reduced and alkylated A chains. A, B1, B2 and C polypeptides are all glycosylated by an intracellular process involving the addition of tunicamycin and endo-beta-N-acetylglucosaminidase-H-sensitive N-linked oligosaccharide side chains. Further glycosylation probably occurs around the time of secretion. Disulphide bonding of some A and B chains can be observed in the cytoplasm within 10 min of adding [35S]methionine. However, it appears that some free A and B2 chains are present in the cytoplasm and that free A chains exist in the medium. The relationship between the 150 x 10(3)-Mr C glycoprotein and the A and B components is discussed. Although B and C chains generate different peptide maps after digestion with S. aureus V8 protease, antibodies raised against isolated, denatured C chains cross-react with reduced and alkylated B (but not A) chains. This suggests that B and C chains may share some antigenic determinant(s).
利用[35S]甲硫氨酸标记小鼠壁内胚层细胞，研究了细胞外基质蛋白层粘连蛋白的多肽A（Mr = 450×10(3)）、B1（Mr = 240×10(3)）、B2（Mr = 230×10(3)）和C（Mr = 150×10(3)）的生物合成及加工过程。多种证据表明，A链并非较小B链的前体。首先，脉冲追踪实验显示，细胞质中A链和（B1 + B2）链的放射性以相同的半衰期（约70分钟）下降。其次，用金黄色葡萄球菌V8蛋白酶消化A链和B（B1 + B2）链所产生的肽图不同。最后，针对分离的、变性的（B1 + B2）链制备的兔抗体与还原并烷基化的A链不发生交叉反应。A、B1、B2和C多肽均通过细胞内过程进行糖基化，该过程涉及添加衣霉素及对endo-β-N-乙酰葡糖胺糖苷酶H敏感的N-连接寡糖侧链。进一步的糖基化可能在分泌时发生。在添加[35S]甲硫氨酸后10分钟内，可在细胞质中观察到一些A链和B链形成二硫键。然而，似乎细胞质中存在一些游离的A链和B2链，且培养基中存在游离的A链。文中讨论了150×10(3)-Mr C糖蛋白与A和B组分之间的关系。虽然用金黄色葡萄球菌V8蛋白酶消化后，B链和C链产生不同的肽图，但针对分离的、变性的C链制备的抗体与还原并烷基化的B链（而非A链）发生交叉反应。这表明B链和C链可能共享某些抗原决定簇。
