Uitto J, Lichtenstein J R, Bauer E A
Biochemistry. 1976 Nov 2;15(22):4935-42. doi: 10.1021/bi00667a029.
The genetic type and molecular structure of the precursor forms of collagen synthesized by matrix-free tendon cells isolated from 17-day old chick embryos were examined by chromatographic and electrophoretic techniques. The [14C]proline-labeled collagenous proteins secreted by the cells resolved on diethylaminoethylcellulose into two peaks, A and B. Both peaks contained type I collagenous proteins since on chromatography on carboxymethylcellulose, after limited pepsin proteolysis, both peaks contained alpha1 and alpha2 chains of collagen in a 2:1 ratio, and cyanogen bromide peptide maps of the 14C-labeled protein in both peaks were similar to cyanogen bromide peptide maps derived from authentic type I collagen. Enzymatic digestion with purified mammalian collagenase demonstrated that the collagen precursor in peak B contained noncollagenous peptide extensions at both the amino- and carboxy-terminal ends of the molecule, while peak A had only carboxy-terminal extension peptides. Although both the amino- and carboxy-terminal extensions incorporated radioactive cystine, only the carboxy-terminal extensions contained interchain disulfide bonds. The carboxy-terminal extensions were also shown to incorporate radioactive tryptophan. Since most of the precursor forms of collagen recovered in the incubation medium chromatographed in peak B, it is concluded that matrix-free tendon cells secrete only type I procollagen with extension peptides at both the amino- and carboxy-terminal ends of the molecule.
采用色谱和电泳技术，对从17日龄鸡胚分离的无基质肌腱细胞合成的胶原蛋白前体形式的基因类型和分子结构进行了检测。细胞分泌的[14C]脯氨酸标记的胶原蛋白在二乙氨基乙基纤维素上分离为两个峰，A和B。两个峰均含有I型胶原蛋白，因为在羧甲基纤维素色谱上，经有限的胃蛋白酶水解后，两个峰均含有比例为2:1的胶原蛋白α1和α2链，且两个峰中14C标记蛋白的溴化氰肽图谱与源自 authentic I型胶原蛋白的溴化氰肽图谱相似。用纯化的哺乳动物胶原酶进行酶消化表明，峰B中的胶原蛋白前体在分子的氨基和羧基末端均含有非胶原蛋白肽延伸，而峰A仅具有羧基末端延伸肽。虽然氨基和羧基末端延伸均掺入了放射性胱氨酸，但只有羧基末端延伸含有链间二硫键。羧基末端延伸也显示掺入了放射性色氨酸。由于在孵育培养基中回收的大多数胶原蛋白前体形式在峰B中进行色谱分离，因此得出结论，无基质肌腱细胞仅分泌在分子的氨基和羧基末端均带有延伸肽的I型前胶原。
