Sodek J, Mandell S M
Biochemistry. 1982 Apr 27;21(9):2011-5. doi: 10.1021/bi00538a006.
Collagen synthesis and deposition in the predentine of the continuously erupting rat incisor was analyzed in vivo following a single intraperitoneal injection of [14C]glycine. Newly synthesized collagen was extracted from the dissected predentine with a solution of 1.0 m sodium chloride containing proteolytic enzyme inhibitors. Mature, cross-linked collagen was solubilized by limited pepsin digestion following extractions with 0.5 M acetic acid or 0.1 M penicillamine. Analysis of the radiolabeled collagens by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and fluorography showed that over 97% of collagen synthesized migrated in the alpha 1 (I) and alpha 2 positions following pepsin digestion; the remaining 3% migrated in the positions of alpha 1 (V) and alpha 3 (V) collagens. However, from the ratio of the alpha 1 (I): alpha 2 it was estimated that approximately 30% of the salt-extractable collagen was alpha 1 (I) trimer. The presence of this collagen was confirmed by salt-fractionation and cyanogen bromide digestion patterns. Type I, alpha 1 (I) trimer, and type V collagens were also found in the salt-insoluble tissue residue. In this fraction the alpha 1 (I) trimer comprised 10-15% of the collagen measured as radioactivity but was difficult to discern colorimetrically. Type III collagen could not be detected in any of the fractions analyzed. From the profiles of isotope incorporation into collagens and collagen precursors, it was evident that collagen synthesis and processing was rapid. Processing of type I collagen and probably also alpha 1 (I) trimer proceeded almost entirely through procollagen intermediates. Rapid maturation of the types I and V collagens in the salt-insoluble fraction and the appearance of beta and gamma chains as early as 30 min after isotope administration. Radiolabeled procollagens were also extracted with acetic acid and penicillamine, indicating that cross-linking of collagen precursors may be involved in fiber formation.
在连续萌出的大鼠切牙的前期牙本质中，胶原蛋白的合成与沉积在腹腔单次注射[14C]甘氨酸后进行了体内分析。用含有蛋白水解酶抑制剂的1.0 m氯化钠溶液从解剖的前期牙本质中提取新合成的胶原蛋白。成熟的、交联的胶原蛋白在用0.5 M乙酸或0.1 M青霉胺提取后，通过有限的胃蛋白酶消化使其溶解。通过十二烷基硫酸钠-聚丙烯酰胺凝胶电泳和荧光自显影对放射性标记的胶原蛋白进行分析表明，胃蛋白酶消化后，超过97%合成的胶原蛋白在α1（I）和α2位置迁移；其余3%在α1（V）和α3（V）胶原蛋白的位置迁移。然而，根据α1（I）:α2的比例估计，约30%的盐可提取胶原蛋白是α1（I）三聚体。这种胶原蛋白的存在通过盐分级和溴化氰消化模式得到证实。I型、α1（I）三聚体和V型胶原蛋白也存在于盐不溶性组织残渣中。在该部分中，α1（I）三聚体占以放射性测量的胶原蛋白的10 - 15%，但用比色法难以辨别。在分析的任何部分中均未检测到III型胶原蛋白。从同位素掺入胶原蛋白和胶原蛋白前体的情况来看，很明显胶原蛋白的合成和加工过程很快。I型胶原蛋白以及可能还有α1（I）三聚体的加工几乎完全通过前胶原中间体进行。盐不溶性部分中I型和V型胶原蛋白的快速成熟以及在同位素给药后30分钟就出现了β链和γ链。放射性标记的前胶原也用乙酸和青霉胺提取，这表明胶原蛋白前体的交联可能参与纤维形成。
