Stassen F L, Cardinale G J, Udenfriend S
Proc Natl Acad Sci U S A. 1973 Apr;70(4):1090-3. doi: 10.1073/pnas.70.4.1090.
The addition of ascorbate to the culture medium of early log-phase mouse fibroblasts (L-929 cells) resulted in a 5-fold increase of prolyl hydroxylase activity. Maximal activity was reached within 2 hr after addition of 5 muM ascorbate. The total amount of enzyme-related antigen did not change on treatment with ascorbate and the activation was shown to be independent of RNA and protein synthesis. The increase in activity caused by ascorbate is therefore due to the activation of a preformed precursor. Enzyme (molecular weight 260,000-300,000) and putative precursor (molecular weight 85,000-105,000) were separated by chromatography on DEAE-Sephadex. Treatment of intact cells with dithiothreitol resulted in an almost quantitative conversion of the enzyme to the smaller inactive protein. When these cells were treated with ascorbate or incubated overnight in fresh medium the enzyme reappeared and precursor concentrations decreased proportionately. Ascorbate may act by bringing about aggregation of enzymatically inactive subunits.
在对数生长期早期的小鼠成纤维细胞（L-929细胞）培养基中添加抗坏血酸盐，导致脯氨酰羟化酶活性增加了5倍。添加5μM抗坏血酸盐后2小时内达到最大活性。用抗坏血酸盐处理后，酶相关抗原的总量没有变化，并且显示这种激活与RNA和蛋白质合成无关。因此，抗坏血酸盐引起的活性增加是由于预先形成的前体的激活。通过在DEAE-葡聚糖凝胶上进行色谱分离，将酶（分子量260,000 - 300,000）和假定的前体（分子量85,000 - 105,000）分开。用二硫苏糖醇处理完整细胞导致酶几乎定量地转化为较小的无活性蛋白。当用抗坏血酸盐处理这些细胞或在新鲜培养基中孵育过夜时，酶重新出现，前体浓度相应降低。抗坏血酸盐可能通过使无酶活性的亚基聚集而起作用。
