Formation of collagen fibrils in vitro by cleavage of procollagen with procollagen proteinases.
Formation of collagen fibrils in vitro by cleavage of procollagen with procollagen proteinases.
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DOI：
10.1016/s0021-9258(18)34351-5
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发表时间：
1982-07
期刊：
影响因子：
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通讯作者：
Michinori Miyahara;Francois;Njieha;Darwin;Prockop
中科院分区：
文献类型：
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作者：
Michinori Miyahara;Francois;Njieha;Darwin;Prockop
建立了一种新的体外研究胶原纤维组装的系统。使用含有前胶原N-蛋白酶和C-蛋白酶活性的部分纯化的酶制剂（EC 3.4.24.00），通过在35-37 ℃的生理缓冲液中从I型前胶原中除去N-和C-前肽来引发原纤维形成。原纤维形成的动力学与在相同的缓冲系统中用组织提取的胶原蛋白形成原纤维所观察到的动力学相似，除了滞后期更长。较长的滞后期部分是由前胶原转化为胶原所需的时间造成的。当使用含有C-前肽但不含N-前肽的中间体作为底物时，获得了类似的结果。因此，在本文所用的条件下，去除c-前肽似乎是原纤维形成的关键步骤。在显微镜下，原胶原或pCcollagen酶裂解形成的原纤维比在相同条件下直接由胶原形成的原纤维更紧密。通过观察到由原胶原裂解形成的原纤维比在相同条件下由提取的胶原形成的纤维在高1.5-2 ℃的温度下稳定，证实了这一印象。当使用较少量的前胶原蛋白酶时，前胶原裂解成胶原的速率显著降低。在这些条件下形成的原纤维的直径高达3微米。有些似乎含有分支点。
A new system was developed for studying the assembly of collagen fibrils in vitro. A partially purified enzyme preparation containing both procollagen N-proteinase and c-proteinase (EC 3.4.24.00) activities was used to initiate fibril formation by removal of the N- and C-propeptides from type I procollagen in a physiological buffer at 35-37 degrees C. The kinetics of fibril formation were similar to those observed for fibril formation with tissue-extracted collagen in the same buffer system, except that the lag phase was longer. The longer lag phase was in part accounted for by the time required to convert procollagen to collagen. Similar results were obtained when an intermediate containing the C-propeptide but not the N-propeptide was used as a substrate. Therefore, removal of the c-propeptide appeared to be the critical step for fibril formation under the conditions used here. The fibrils formed by enzymic cleavage of procollagen or pCcollagen appeared microscopically to be more tightly packed than fibrils formed directly from collagen under the same conditions. This impression was confirmed by the observation that the fibrils formed by cleavage of procollagen were stable to temperatures 1.5-2 degrees C higher than fibers formed from extracted collagen under the same conditions. When smaller amounts of procollagen proteinase were used, the rate of cleavage of procollagen to collagen was markedly reduced. The fibrils which formed under these conditions were up to 3 micrometers in diameter. Some appeared to contain branch points.