Uitto J, Booth B A, Polak K L
Biochim Biophys Acta. 1980 Aug 21;624(2):545-61. doi: 10.1016/0005-2795(80)90095-1.
Human skin fibroblasts in culture have previously been shown to synthesize genetically distinct procollagens type I and type III. In the present study, cultured human skin fibroblasts were incubated under conditions optimized for synthesis of these procollagens in medium containing [3H]proline. The newly synthesized type I and type III 3H-labeled procollagens in the culture medium were then isolated as native proteins by DEAE-cellulose chromatography, or by gel filtration and SDS-polyacrylamide slab gel electrophoresis under denaturing conditons after limited pepsin proteolysis. The chromatographic procedures were optimized to yield reliable and reporducible results with good recoveries. The isolated procollagens were identified by cyanogen bromide peptide mapping and characterized by cleavage with highly purified collagenase synthesized by human skin fibroblasts. Assay of the relative synthesis of type I/III procollagens by normal human skin fibroblasts using DEAE-cellulose chromatography indicated that 80% of the procollagen in the medium was type I while the remaining 20% consisted of type III. When the ratio of newly-synthesized type I/III collagens was estimated by gel filtration or using SDS-polyacrylamide slab gel electrophoresis after limited pepsin proteolysis, relatively fewer type III collagen alpha-chains were recovered. This observation suggests that some of the type of the type III collagen molecules are in a conformation which is less resistant to digestion by pepsin than the triple-helix of type I procollagen. The coefficient of variation for the relative synthesis of type I and type III procollagens by control cultures was relatively small (16%), indicating that the phenotypic expression of type I and type III procollagen genes, under optimized culture conditions, is under a relatively tight control. The results further suggest that the optimized methodology developed for assay of the relative synthesis of type I and type III procollagens and collagens by cultured human skin fibroblasts can be utilized in studies on collagen aberrations in acquired and inherited diseases of connective tissue.
先前已证明，培养的人皮肤成纤维细胞能合成基因不同的I型和III型前胶原。在本研究中，将培养的人皮肤成纤维细胞在含[3H]脯氨酸的培养基中，于优化的有利于这些前胶原合成的条件下孵育。然后，通过DEAE - 纤维素色谱法，或在有限胃蛋白酶水解后，经凝胶过滤以及在变性条件下进行SDS - 聚丙烯酰胺平板凝胶电泳，将培养基中新合成的I型和III型3H标记前胶原作为天然蛋白质分离出来。对色谱程序进行了优化，以产生可靠且可重复的结果，并具有良好的回收率。通过溴化氰肽图谱鉴定分离出的前胶原，并用由人皮肤成纤维细胞合成的高度纯化的胶原酶切割进行表征。使用DEAE - 纤维素色谱法对正常人皮肤成纤维细胞I型/III型前胶原的相对合成进行测定，结果表明培养基中80%的前胶原为I型，其余20%为III型。当通过凝胶过滤或在有限胃蛋白酶水解后使用SDS - 聚丙烯酰胺平板凝胶电泳来估计新合成的I型/III型胶原的比例时，回收的III型胶原α链相对较少。这一观察结果表明，某些III型胶原分子的构象比I型前胶原的三螺旋结构对胃蛋白酶消化的抵抗力更弱。对照培养物中I型和III型前胶原相对合成的变异系数相对较小（16%），这表明在优化的培养条件下，I型和III型前胶原基因的表型表达受到相对严格的控制。结果还表明，所开发的用于测定培养的人皮肤成纤维细胞I型和III型前胶原及胶原相对合成的优化方法，可用于研究结缔组织获得性和遗传性疾病中的胶原异常。
