Identification of specific proteins synthesized by type II pneumocytes in primary culture.
Identification of specific proteins synthesized by type II pneumocytes in primary culture.
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鉴定原代培养物中 II 型肺细胞合成的特定蛋白质。
DOI：
10.1042/bj2100607
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发表时间：
1983
期刊：
影响因子：
--
通讯作者：
Rannels,DE
中科院分区：
文献类型：
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作者：
Wartell,SA;Nafe,CK;Watkins,CA;Rannels,DE
十二烷基硫酸钠/聚丙烯酰胺凝胶电泳，以确定II型细胞特异性蛋白质的蛋白质，从第二型颗粒肺细胞的原代培养物进行了检查。比较了考马斯亮蓝染色带在细胞蛋白、培养液、灌洗液和板层体中的分布。最突出的染色带在无血清培养基中的II型细胞培养物（HS 1; Mr 39900）对应于一个主要的蛋白质在无细胞的可沉淀（20000克，30分钟）粗表面活性剂从大鼠肺通过生理盐水（0.9%NaCl）灌洗。第二种蛋白质（HS 2; Mr 12000）也发现在II型细胞条件培养基和灌洗。大鼠血清和供体小牛血清（用于分离II型细胞）均不含与HS 1或HS 2蛋白共迁移的蛋白质。HS 1也存在于细胞蛋白和从整个肺分离的板层体的考马斯蓝染色凝胶中。如培养基的十二烷基硫酸钠/聚丙烯酰胺凝胶的放射自显影所示，II型细胞的培养物将[14 C]苯丙氨酸掺入HS 1和HS 2中。原位灌注的大鼠肺将[35 S]甲硫氨酸掺入板层体组分中的HS 1中。第三种蛋白质（HS 3; Mr 47000）仅在细胞培养基的放射自显影中观察到;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。在来自肺泡巨噬细胞、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对应于HS 1、HS 2或HS 3的蛋白条带。HS 1的二维凝胶电泳显示等电点为6.3的单一多肽; HS 3显示为等电点范围为6.3至6.6的斑点链。HS 2尚未在二维凝胶上鉴定。HS 1的氨基酸组成与以前研究的表面活性剂脱辅基蛋白没有显着差异;然而，HS 1未被糖蛋白染色剂检测到，也没有出现巯基连接的多聚体的亚基。
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.