Wartell S A, Nafe C K, Watkins C A, Rannels D E
Biochem J. 1983 Feb 15;210(2):607-16. doi: 10.1042/bj2100607.
Proteins from primary cultures of type II granular pneumocytes have been examined by sodium dodecyl sulphate/polyacrylamide-gel electrophoresis to identify type II cell-specific proteins. The distribution of Coomassie Blue-stained bands in preparations of cellular proteins, culture medium, lavage and lamellar bodies have been compared. The most prominent stained band in the serum-free medium from type II cell cultures (HS1; Mr 39900) corresponds to a major protein in acellular sedimentable (20000 g for 30 min) crude surfactant obtained from rat lungs by saline (0.9% NaCl) lavage. A second protein (HS2; Mr 12000) is also found both in type II cell-conditioned medium and in lavage. Neither rat serum nor donor calf serum (used in the isolation of the type II cells) contains a protein co-migrating with HS1 or HS2 proteins. HS1 is also found in Coomassie Blue-stained gels of cellular proteins and of lamellar bodies isolated from whole lungs. Cultures of type II cells incorporate [14C]phenylalanine into HS1 and HS2 as shown by autoradiography of sodium dodecyl sulphate/polyacrylamide gels of culture medium. Rat lungs perfused in situ incorporate [35S]methionine into HS1 in the lamellar body fraction. A third protein (HS3; Mr 47000) is observed only in autoradiographs of cell culture medium; no corresponding Coomassie Blue-stained band can be identified in medium, in cells or in lung lavage. No protein bands corresponding to HS1, HS2 or HS3 are found in conditioned media from pulmonary alveolar macrophages, rat fibroblasts or bovine aorta endothelial cells. Two-dimensional gel electrophoresis of HS1 shows a single polypeptide with an isoelectric point of 6.3; HS3 appears as a chain of spots with a range of isoelectric points from 6.3 to 6.6. HS2 has not been identified on two-dimensional gels. The amino acid composition of HS1 does not differ significantly from that of surfactant apoproteins studied previously; however, HS1 is not detected by glycoprotein stains, nor does it appear to be a subunit of a thiol-linked multimer.
通过十二烷基硫酸钠/聚丙烯酰胺凝胶电泳对II型颗粒肺细胞原代培养物中的蛋白质进行了检测，以鉴定II型细胞特异性蛋白质。比较了细胞蛋白质制剂、培养基、灌洗液和板层小体中考马斯亮蓝染色条带的分布。II型细胞培养物的无血清培养基中最显著的染色条带（HS1；分子量39900）对应于通过盐水（0.9%氯化钠）灌洗从大鼠肺中获得的无细胞可沉淀（20000g离心30分钟）粗表面活性物质中的一种主要蛋白质。第二种蛋白质（HS2；分子量12000）在II型细胞条件培养基和灌洗液中也有发现。大鼠血清和供体小牛血清（用于分离II型细胞）中均不含有与HS1或HS2蛋白共迁移的蛋白质。在全肺分离的细胞蛋白质和板层小体的考马斯亮蓝染色凝胶中也发现了HS1。如培养基的十二烷基硫酸钠/聚丙烯酰胺凝胶放射自显影所示，II型细胞培养物将[14C]苯丙氨酸掺入HS1和HS2中。原位灌注的大鼠肺将[35S]甲硫氨酸掺入板层小体部分的HS1中。第三种蛋白质（HS3；分子量47000）仅在细胞培养基的放射自显影片中观察到；在培养基、细胞或肺灌洗液中未发现相应的考马斯亮蓝染色条带。在肺泡巨噬细胞、大鼠成纤维细胞或牛主动脉内皮细胞的条件培养基中未发现与HS1、HS2或HS3对应的蛋白条带。HS1的二维凝胶电泳显示一条单一多肽，其等电点为6.3；HS3表现为一系列等电点在6.3至6.6之间的斑点链。HS2在二维凝胶上未被鉴定。HS1的氨基酸组成与先前研究的表面活性物质载脂蛋白没有显著差异；然而，糖蛋白染色未检测到HS1，它似乎也不是硫醇连接的多聚体的亚基。
