Rowe L B, Schwarz R I
Mol Cell Biol. 1983 Feb;3(2):241-9. doi: 10.1128/mcb.3.2.241-249.1983.
Two factors must be present for primary avian tendon cells to commit 50% of their total protein production to procollagen: ascorbate and high cell density. Scorbutic primary avian tendon cells at high cell density (greater than 4 X 10(4) cells per cm2) responded to the addition of ascorbate by a sixfold increase in the rate of procollagen synthesis. The kinetics were biphasic, showing a slow increase during the first 12 h followed by a more rapid rise to a maximum after 36 to 48 h. In contrast, after ascorbate addition, the level of accumulated cytoplasmic procollagen mRNA (alpha 2) showed a 12-h lag followed by a slow linear increase requiring 60 to 72 h to reach full induction. At all stages of the induction process, the relative increase in the rate of procollagen synthesis over the uninduced state exceeded the relative increase in the accumulation of procollagen mRNA. A similar delay in mRNA induction was observed when the cells were grown in an ascorbate-containing medium but the cell density was allowed to increase. In all cases, the rate of procollagen synthesis peaked approximately 24 h before the maximum accumulation of procollagen mRNA. The kinetics for the increase in procollagen synthesis are not, therefore, in agreement with the simple model that mRNA levels are the rate-limiting factor in the collagen pathway. We propose that the primary control point is at a later step. Further support for this idea comes from inhibitor studies, using alpha, alpha'-dipyridyl to block ascorbate action. In the presence of 0.3 mM alpha, alpha'-dipyridyl there was a specific two- to threefold decrease in procollagen production after 4 h, but this was unaccompanied by a drop in procollagen mRNA levels. Therefore, inhibitor studies give further support to the idea that primary action of ascorbate is to release a post-translational block.
原代禽腱细胞要将其总蛋白产量的50%用于合成前胶原，必须具备两个因素：抗坏血酸和高细胞密度。处于高细胞密度（每平方厘米大于4×10⁴个细胞）的坏血病原代禽腱细胞，在添加抗坏血酸后，前胶原合成速率增加了六倍。其动力学是双相的，在最初12小时内缓慢增加，随后在36至48小时后更迅速地上升至最大值。相比之下，添加抗坏血酸后，积累的细胞质前胶原mRNA（α2）水平出现12小时的延迟，随后缓慢线性增加，需要60至72小时才能达到完全诱导。在诱导过程的所有阶段，前胶原合成速率相对于未诱导状态的相对增加超过了前胶原mRNA积累的相对增加。当细胞在含抗坏血酸的培养基中生长但允许细胞密度增加时，观察到mRNA诱导有类似的延迟。在所有情况下，前胶原合成速率在达到前胶原mRNA最大积累量之前约24小时达到峰值。因此，前胶原合成增加的动力学与mRNA水平是胶原途径中的限速因素这一简单模型不一致。我们提出主要控制点在后期步骤。使用α,α'-联吡啶阻断抗坏血酸作用的抑制剂研究为这一观点提供了进一步支持。在存在0.3 mM α,α'-联吡啶的情况下，4小时后前胶原产量有特定的两到三倍下降，但这并未伴随着前胶原mRNA水平的下降。因此，抑制剂研究进一步支持了抗坏血酸的主要作用是解除翻译后阻断这一观点。
