Macarak E J
J Cell Physiol. 1984 May;119(2):175-82. doi: 10.1002/jcp.1041190206.
Pulmonary artery endothelial cells were isolated from bovine fetal blood vessels and used for biosynthetic studies. At confluence, cultures were incubated in minimal essential medium (MEM) without serum containing [U-14C]proline. After 24 hours, medium was removed and labeled proteins were precipitated by the addition of ammonium sulfate and fractionated by diethylaminoethyl (DEAE)-cellulose chromatography. The elution profile showed four major peaks and one minor peak. Fractions within each peak were pooled, subjected to digestion by chymotrypsin and/or collagenase, and analyzed by polyacrylamide gel electrophoresis. Peak l contained a collagen which contained approximately 6% of the 3-hydroxyproline isomer while total hydroxyproline content was approximately 45%. This material was digested by purified bacterial collagenase and had a mobility slightly slower than that of alpha 1(III) which did not change under conditions that reduce disulfide bonds. Upon digestion with chymotrypsin under conditions where native procollagens are converted to alpha-chains, this material was digested. These properties suggest that this material is type VIII or EC (endothelial cell) collagen. Peak 2 contained substantial fibronectin while peak 3 contained primarily type III procollagen. The last major peak contained a mixture of collagenous and noncollagenous material. Upon digestion with chymotrypsin, several peptides were generated which were sensitive to bacterial collagenases. The two major chymotrypsin-resistant components had mobilities slower than that of alpha(III) and were not disulfide-bonded.
从牛胎儿血管中分离出肺动脉内皮细胞，并用于生物合成研究。细胞汇合后，将培养物在不含血清但含有[U-14C]脯氨酸的最低限度基本培养基（MEM）中孵育。24小时后，去除培养基，通过添加硫酸铵沉淀标记的蛋白质，并通过二乙氨基乙基（DEAE）-纤维素色谱法进行分级分离。洗脱图谱显示有四个主要峰和一个次要峰。每个峰内的组分合并，用胰凝乳蛋白酶和/或胶原酶消化，然后通过聚丙烯酰胺凝胶电泳进行分析。峰1含有一种胶原蛋白，其中3-羟脯氨酸异构体约占6%，而总羟脯氨酸含量约为45%。该物质被纯化的细菌胶原酶消化，其迁移率略慢于α1(III)，在还原二硫键的条件下其迁移率不变。在天然前胶原转化为α链的条件下用胰凝乳蛋白酶消化时，该物质被消化。这些特性表明该物质是VIII型或内皮细胞（EC）胶原蛋白。峰2含有大量纤连蛋白，而峰3主要含有III型前胶原。最后一个主要峰含有胶原性和非胶原性物质的混合物。用胰凝乳蛋白酶消化后，产生了几种对细菌胶原酶敏感的肽。两种主要的抗胰凝乳蛋白酶组分的迁移率比α(III)慢，且不是通过二硫键结合的。
