Miyahara M, Hayashi K, Berger J, Tanzawa K, Njieha F K, Trelstad R L, Prockop D J
J Biol Chem. 1984 Aug 10;259(15):9891-8.
Two systems were used to generate collagen fibrils in vitro by enzymic cleavage of intermediates in the conversion of procollagen to collagen. In one system fibrils were generated by using procollagen NH2-terminal proteinase to cleave pNcollagen, the intermediate which contains the NH2-terminal but not the COOH-terminal propeptides found in procollagen. When pNcollagen was incubated with procollagen NH2-terminal proteinase, the NH2-terminal propeptides were enzymically cleaved from the protein, and there was an increase in the turbidity of the solution over and above the turbidity observed with pNcollagen alone. Electron microscope examination of the samples demonstrated that the increase in turbidity was associated with the assembly of collagen fibrils. The fibrils had a mean diameter of 104 nm +/- 51.7 S.D. or about the same as fibrils formed from pNcollagen alone. However, the fibrils formed by enzymic cleavage of pNcollagen had a more distinct gap-overlap pattern and they appeared to be more tightly packed than fibrils of pNcollagen. Varying the concentration of enzyme varied both the rate of enzymic cleavage of the pNcollagen and the rate of fibril assembly, but there was no consistent effect on the diameter or morphology of the fibrils. In the second system, fibrils were generated with a recently described procedure (Miyahara, M., Njieha, F. K., and Prockop, D. J. (1982) J. Biol. Chem. 257, 8442-8448) in which procollagen COOH-terminal proteinase is used to cleave pCcollagen, the intermediate containing the COOH-terminal but not the NH2-terminal propeptides found in procollagen. When incubated with procollagen COOH-terminal proteinase, the COOH-terminal propeptides were cleaved and collagen fibrils assembled. The collagen fibrils were unusually thick with a mean diameter of 1184 nm +/- 291 S.D. The large diameters of the fibrils made it possible to demonstrate by scanning electron microscopy that each fibril was comprised of a bundle of subfibrils packed into a right-handed helix. The fibrils frequently had branch points which appeared to consist of subfibrils which separated from the main axis of the structure. Also, the surface of the fibrils was scalloped at 270- to 300-nm intervals, suggesting that some of the collagen molecules on the surface were in a 4D staggered array. The results suggested the hypothesis that the order in which the NH2-terminal and COOH-terminal propeptides are cleaved in the conversion of procollagen to collagen may provide a mechanism for controlling the diameter, or both the diameter and morphology, of collagen fibrils.
通过酶解原胶原转化为胶原过程中的中间体，在体外生成胶原纤维时使用了两种系统。在一种系统中，通过使用原胶原氨基端蛋白酶切割前胶原（pNcollagen）来生成纤维，前胶原是一种中间体，它含有原胶原中的氨基端但不含有羧基端前肽。当将前胶原与原胶原氨基端蛋白酶一起温育时，氨基端前肽会被酶从蛋白质上切割下来，并且溶液的浊度会比单独使用前胶原时观察到的浊度有所增加。对样品进行电子显微镜检查表明，浊度的增加与胶原纤维的组装有关。这些纤维的平均直径为104纳米±51.7标准差，与仅由前胶原形成的纤维直径大致相同。然而，通过酶解前胶原形成的纤维具有更明显的间隙 - 重叠模式，并且它们看起来比前胶原纤维堆积得更紧密。改变酶的浓度会同时改变前胶原的酶解速率和纤维组装速率，但对纤维的直径或形态没有一致的影响。在第二个系统中，使用最近描述的方法（宫原，M.，恩杰哈，F.K.，和普罗科普，D.J.（1982年）《生物化学杂志》257，8442 - 8448）生成纤维，其中使用原胶原羧基端蛋白酶切割前胶原（pCcollagen），前胶原是一种中间体，它含有原胶原中的羧基端但不含有氨基端前肽。当与原胶原羧基端蛋白酶一起温育时，羧基端前肽被切割，胶原纤维组装。这些胶原纤维异常粗大，平均直径为1184纳米±291标准差。纤维的大直径使得通过扫描电子显微镜能够证明每个纤维由一束亚纤维组成，这些亚纤维堆积成右手螺旋。这些纤维经常有分支点，这些分支点似乎由从结构主轴分离的亚纤维组成。此外，纤维表面以270至300纳米的间隔呈扇形，这表明表面上的一些胶原分子呈4D交错排列。结果提出了一个假设，即在原胶原转化为胶原的过程中，氨基端和羧基端前肽的切割顺序可能为控制胶原纤维的直径或直径与形态两者提供一种机制。
