Church R L
Invest Ophthalmol Vis Sci. 1980 Feb;19(2):192-202.
Procollagen and collagen were isolated from the culture medium of normal bovine corneal stromal fibroblasts. DEAE-cellulose chromatography was used to separate the collagen molecules from the different procollagens present. One collagen and four procollagen peaks were isolated and biochemically characterized. All the procollagen fractions and the collagen fraction yielded, after limited pepsin or chymotrypsin digestion followed by CNBr digestion, molecules that correspond to (alpha 1)2 alpha 2 exclusively. Thus only type I collagen is found in the growth medium of of bovine corneal stromal fibroblast cultures. Each of the individual procollagen peaks contained pro-alpha chains having molecular weights of 120,000, 150,000, 165,000, 180,000, and 190,000 daltons, according to their elution position on DEAE-cellulose. The presence of type I procollagen molecules having pro-alpha chains of 165,000, 180,000, and 190,000 daltons has not previously been reported and probably represents higher-molecular-weight precursor intermediates. The amino acid compositions of the different procollagen fractions are unique, and each contains relatively large amounts of cysteine and tryptophan. Carbohydrate analysis, cyanogen bromide peptide analysis, electron microscopy of SLS-crystallities, and SDS-polyacrylamide gel electrophoresis were used to further characterize the procollagen and collagen molecules.
从正常牛角膜基质成纤维细胞的培养基中分离出前胶原和胶原。采用二乙氨基乙基纤维素色谱法从存在的不同前胶原中分离胶原分子。分离出一个胶原峰和四个前胶原峰，并进行了生化特性鉴定。所有前胶原组分和胶原组分在经有限的胃蛋白酶或胰凝乳蛋白酶消化后再经溴化氰消化，产生的分子仅对应于(α1)2α2。因此，在牛角膜基质成纤维细胞培养物的生长培养基中仅发现I型胶原。根据各个前胶原峰在二乙氨基乙基纤维素上的洗脱位置，每个峰都含有分子量为120,000、150,000、165,000、180,000和190,000道尔顿的前α链。以前尚未报道过具有165,000、180,000和190,000道尔顿前α链的I型前胶原分子的存在，它们可能代表更高分子量的前体中间体。不同前胶原组分的氨基酸组成是独特的，并且每种都含有相对大量的半胱氨酸和色氨酸。采用碳水化合物分析、溴化氰肽分析、十二烷基硫酸钠-结晶性纤维状蛋白的电子显微镜检查以及十二烷基硫酸钠-聚丙烯酰胺凝胶电泳对前胶原和胶原分子进行进一步鉴定。
