Bellon G, Monboisse J C, Randoux A, Borel J P
Biochim Biophys Acta. 1987 Aug 19;930(1):39-47. doi: 10.1016/0167-4889(87)90153-4.
A technique of derivatizing proline and 4-hydroxyproline with 7-chloro-4-nitrobenzo-2-oxa-1,3-diazole was used to measure the radioactivities, concentrations and specific activities of proline and hydroxyproline. The technique was used to study the conditions of procollagen synthesis in cultured human foreskin fibroblasts. Procollagen synthesis appeared to be independent of the proline concentration in the medium, in the presence of glutamine, when monitored by the assay of non-dialyzable hydroxyproline, but not when monitored by [14C]proline incorporation. In the absence of unlabelled proline added to labelled proline in the medium, the specific activity of the secreted procollagen did not reach a plateau over a 24-h period. When the medium was supplemented with glutamine, glutamic acid, or aspartic acid, both the radioactivity and concentration of intracellular free proline decreased. Pyrrolidone-2-carboxylic acid and ornithine both induced a slight increase in concentration of the intracellular free proline. Glutamine competed with [14C]proline for incorporation into prolyl-tRNA and procollagen, independently of free intracellular proline, and it stimulated the biosynthesis of procollagen (expressed as non-dialyzable hydroxyproline) by a factor of 2.3.
采用一种用7-氯-4-硝基苯并-2-恶唑-1,3-二唑衍生化脯氨酸和4-羟脯氨酸的技术来测量脯氨酸和羟脯氨酸的放射性、浓度及比活性。该技术用于研究培养的人包皮成纤维细胞中前胶原合成的条件。当通过不可透析羟脯氨酸测定进行监测时，在前胶原合成过程中，谷氨酰胺存在的情况下，前胶原合成似乎与培养基中的脯氨酸浓度无关；但当通过[14C]脯氨酸掺入进行监测时并非如此。在培养基中未向标记脯氨酸添加未标记脯氨酸的情况下，分泌的前胶原的比活性在24小时内未达到平稳状态。当培养基补充谷氨酰胺、谷氨酸或天冬氨酸时，细胞内游离脯氨酸的放射性和浓度均降低。吡咯烷-2-羧酸和鸟氨酸均诱导细胞内游离脯氨酸浓度略有增加。谷氨酰胺与[14C]脯氨酸竞争掺入脯氨酰-tRNA和前胶原，这与细胞内游离脯氨酸无关，并且它将前胶原的生物合成（以不可透析羟脯氨酸表示）刺激了2.3倍。
