Assembly of collagen fibrils de novo by cleavage of the type I pC-collagen with procollagen C-proteinase. Assay of critical concentration demonstrates that collagen self-assembly is a classical example of an entropy-driven process.
Assembly of collagen fibrils de novo by cleavage of the type I pC-collagen with procollagen C-proteinase. Assay of critical concentration demonstrates that collagen self-assembly is a classical example of an entropy-driven process.
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DOI：
10.1016/s0021-9258(18)47783-6
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发表时间：
1987-11
期刊：
影响因子：
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通讯作者：
K. Kadler;Y. Hojima;D. J. Prockop
中科院分区：
文献类型：
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作者：
K. Kadler;Y. Hojima;D. J. Prockop
用14C标记的氨基酸从培养的人成纤维细胞培养上清液中提纯I型前胶原，用前胶原N-蛋白酶裂解NH2末端的前肽，用凝胶过滤层析分离得到PC-胶原。在34℃的生理缓冲液中，PC-胶原即使在0.5mg.ml-1的浓度下也不能聚集成纤维或大的聚集体。然而，PC-胶原被纯化的C-蛋白酶切割成胶原(Hojima，Y.，(1985)J.Biol.化学。260、15996-16003)产生肉眼可见且大到足以通过13,000xg离心4分钟从溶液中分离的纤维。在高浓度酶作用下，PC-胶原在1h内完全裂解，3h时浑浊度接近最大，但胶原在纤维中持续存在10h以上，由于PC-胶原被均匀地标记在14C-氨基酸上，因此直接确定了可溶胶原的浓度，从而确定了聚合的临界浓度。该临界浓度与PC-胶原的初始浓度和切割速度无关。临界浓度在29~41℃范围内随温度升高而降低，为0.12+/-0.06(S.E.)在41℃时，组装体的热力学参数在29-41℃范围内基本上与温度无关。该过程是吸热的，增量H值为+56kcal.mol-1，但熵驱动的增量S值为+220cal.K-1.mol-1。在37℃时，聚合的吉布斯能量变化为-13kcal.mol-1。数据首次表明，I型胶原纤维从头形成是类似于肌动蛋白、鞭毛和烟草花叶病毒蛋白聚合的熵驱动自组装过程的经典例子。
Type I procollagen was purified from the medium of cultured human fibroblasts incubated with 14C-labeled amino acids, the NH2-terminal propeptides were cleaved with procollagen N-proteinase, and the resulting pC-collagen was isolated by gel filtration chromatography. pC-collagen did not assemble into fibrils or large aggregates even at concentrations of 0.5 mg.ml-1 at 34 degrees C in a physiological buffer. However, cleavage of pC-collagen to collagen with purified C-proteinase (Hojima, Y., (1985) J. Biol. Chem. 260, 15996-16003) generated fibrils that were visible by eye and that were large enough to be separated from solution by centrifugation at 13,000 x g for 4 min. With high concentrations of enzyme, the pC-collagen was completely cleaved in 1 h, and turbidity was near maximal in 3 h, but collagen continued to be incorporated in fibrils for over 10 h. Because the pC-collagen was uniformly labeled with 14C-aminoacids, the concentration of soluble collagen and, therefore, the critical concentration of polymerization were determined directly. The critical concentration was independent of the initial pC-collagen concentration and of the rate of cleavage. The critical concentration decreased with temperature between 29 and 41 degrees C and was 0.12 +/- 0.06 (S.E.) microgram.ml-1 at 41 degrees C. The thermodynamic parameters of assembly were essentially independent of temperature in the range 29 to 41 degrees C. The process was endothermic with a delta H value of +56 kcal.mol-1, but entropy driven with a delta S value of +220 cal.K-1.mol-1. The Gibbs energy change for polymerization was -13 kcal.mol-1 at 37 degrees C. The data demonstrate, for the first time, that type I collagen fibril formation de novo is a classical example of an entropy-driven self-assembly process similar to the polymerization of actin, flagella, and tobacco mosaic virus protein.