Kadler K E, Hojima Y, Prockop D J
Department of Biochemistry and Molecular Biology, Jefferson Medical College, Thomas Jefferson University, Philadelphia, Pennsylvania 19107.
J Biol Chem. 1987 Nov 15;262(32):15696-701.
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.
从用¹⁴C标记氨基酸培养的人成纤维细胞培养基中纯化出I型原胶原，用原胶原N蛋白酶切割其NH₂末端前肽，然后通过凝胶过滤色谱法分离得到的pC-胶原。即使在生理缓冲液中于34℃下浓度达到0.5mg/ml，pC-胶原也不会组装成纤维或大聚集体。然而，用纯化的C蛋白酶（Hojima, Y., (1985) J. Biol. Chem. 260, 15996 - 16003）将pC-胶原切割成胶原后，会产生肉眼可见的纤维，且这些纤维大到足以通过在13,000×g下离心4分钟从溶液中分离出来。在高浓度酶的作用下，pC-胶原在1小时内完全被切割，3小时内浊度接近最大值，但胶原在超过10小时内仍持续掺入纤维中。由于pC-胶原用¹⁴C-氨基酸均匀标记，因此可直接测定可溶性胶原的浓度，进而确定聚合的临界浓度。临界浓度与初始pC-胶原浓度和切割速率无关。在29至41℃之间，临界浓度随温度降低，在41℃时为0.12±0.06（标准误）μg/ml。在29至41℃范围内，组装的热力学参数基本与温度无关。该过程是吸热的，ΔH值为+56 kcal/mol，但由熵驱动，ΔS值为+220 cal·K⁻¹·mol⁻¹。在37℃时，聚合的吉布斯自由能变化为-13 kcal/mol。这些数据首次证明，I型胶原纤维的从头形成是一个熵驱动的自组装过程的经典例子，类似于肌动蛋白、鞭毛和烟草花叶病毒蛋白的聚合。
