Synthesis of prolyl 4-hydroxylase by cultured chicken corneal cells.

Prolyl hydroxylase (EC 1.14.11.2) synthesis was examined in the cultured corneas of 17-day-old chick embryos which were cultured in F-12 medium supplemented with fetal calf serum. The amount of prolyl hydroxylase in cells was measured by immunoelectrophoresis using antibodies directed against this enzyme. The results indicated that the amounts of prolyl hydroxylase depended on cell density. The cells were harvested at the early-log phase and plated at high cell density to mimic the culture condition of cells at the stationary phase (crowding). The amount of 35S-prolyl hydroxylase indicated a two-fold increase in the rate of prolyl hydroxylase synthesis after crowding. By use of 32P-labeled cDNA for the beta-subunit of prolyl hydroxylase a proportionate increase of the mRNA was noted in accordance with the increase in the rate of prolyl hydroxylase synthesis, suggesting that this enzyme synthesis is regulated at the transcriptional level.