Kay E P, Smith R E, Nimni M E
J Biol Chem. 1982 Jun 25;257(12):7116-21.
Corneal endothelial cells in culture synthesize basement membrane collagen and secrete it into the medium. This collagen sediments faster than interstitial collagen by velocity sedimentation and is disulfide-bonded. After reduction, two biochemically distinct chains can be determined by cyanogen bromide peptide mapping. These chains migrate close to each other and immediately below beta 12(I) components on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Treatment with pepsin gives rise to a major band which still retains interchain disulfide bonds and which will not convert to components with the mobility of interstitial alpha chain by reduction. However, an alpha chain and three minor collagenase-sensitive and pepsin-resistant peptides are generated if the molecule is reduced and alkylated under nondenaturing conditions prior to pepsin treatment. When collagen which accumulates in the media over a long period of time is compared to the newly synthesized molecules, there is an apparent differential resistance to limited pepsin treatment. However, the products which are generated in both cases share electrophoretic identity.
培养中的角膜内皮细胞合成基底膜胶原蛋白并将其分泌到培养基中。这种胶原蛋白通过速度沉降比间质胶原蛋白沉降得更快，并且是通过二硫键结合的。还原后，通过溴化氰肽图谱可以确定两条生化性质不同的链。这些链在十二烷基硫酸钠 - 聚丙烯酰胺凝胶电泳上彼此靠近迁移，且紧位于β12（I）组分之下。用胃蛋白酶处理会产生一条主要条带，该条带仍保留链间二硫键，并且不会通过还原转化为具有间质α链迁移率的组分。然而，如果在胃蛋白酶处理之前在非变性条件下将分子还原并烷基化，则会产生一条α链和三个对胶原酶敏感且对胃蛋白酶有抗性的小肽。当将长时间在培养基中积累的胶原蛋白与新合成的分子进行比较时，对有限胃蛋白酶处理存在明显的差异抗性。然而，两种情况下产生的产物具有相同的电泳特性。
