Clark J G, Hilderbran J N
Anal Biochem. 1984 Aug 1;140(2):478-85. doi: 10.1016/0003-2697(84)90197-0.
In vitro procollagen production rates can be determined by culturing cells in the presence of [3H]proline and measuring the subsequent formation of [3H]hydroxyproline. Values of actual procollagen production can be calculated if the total radioactivity and the specific activity of the newly synthesized procollagen is known. A simple microanalytical method for measuring procollagen specific activity in order to determine procollagen production by lung fibroblasts in vitro is reported. Confluent fibroblasts (IMR-90) were cultured in fresh medium containing [3H]proline, and [3H]hydroxyproline production and prolyl hydroxylation were measured. Hydroxyproline specific activity of nondialyzable procollagen in culture medium as well as extracellular and intracellular free proline specific activity were determined by an ultramicromethod in which the radiolabeled amino acids were reacted with [14C]dansyl chloride of known specific activity [Airhart et al. (1979) Anal. Biochem. 96, 45-55]. Procollagen production rates were readily determined by this method using 5 to 20 microCi [3H]proline and approximately 10(6) cells. It was found that 3H-procollagen production rate into culture medium was constant after a lag of 1.6 h, while procollagen production rate (0.23 pmol/microgram DNA . h) was constant from time zero to 9 h. The specific activities of extracellular and intracellular free proline were not constant during the labeling period, nor were they equal to procollagen specific activity. These data indicate that free proline pool specific activities are not a valid measure of procollagen specific activity. The experimental approach described obviates the need to define or characterize the proline precursor pool from which procollagen is synthesized, and may be readily applied to determine fibroblast procollagen production rates in vitro.
体外原胶原生成速率可通过在[3H]脯氨酸存在的情况下培养细胞并测量随后[3H]羟脯氨酸的形成来确定。如果已知新合成原胶原的总放射性和比活性，就可以计算出实际的原胶原生成值。本文报道了一种简单的微量分析方法，用于测量原胶原比活性，从而在体外测定肺成纤维细胞的原胶原生成。将汇合的成纤维细胞（IMR-90）培养在含有[3H]脯氨酸的新鲜培养基中，并测量[3H]羟脯氨酸的生成和脯氨酰羟化作用。通过一种超微量方法测定培养基中不可透析原胶原的羟脯氨酸比活性以及细胞外和细胞内游离脯氨酸的比活性，在该方法中，放射性标记的氨基酸与已知比活性的[14C]丹磺酰氯反应[艾尔哈特等人（1979年）《分析生物化学》96卷，第45 - 55页]。使用5至20微居里的[3H]脯氨酸和约10(6)个细胞，通过该方法可轻松测定原胶原生成速率。结果发现，在1.6小时的延迟后，进入培养基的3H - 原胶原生成速率保持恒定，而原胶原生成速率（0.23皮摩尔/微克DNA·小时）从零小时到9小时保持恒定。在标记期间，细胞外和细胞内游离脯氨酸的比活性并不恒定，也不等于原胶原比活性。这些数据表明，游离脯氨酸池比活性不是原胶原比活性的有效度量。所描述的实验方法无需定义或表征合成原胶原的脯氨酸前体池，并且可轻松应用于体外测定成纤维细胞的原胶原生成速率。
