Related Experiment Video
Updated: Sep 25, 2026
Organotypic Culture Method to Study the Development Of Embryonic Chicken Tissues
Published on: August 25, 2018
Synthesis of prolyl 4-hydroxylase by cultured chicken corneal cells
Insights
Cell density significantly impacts prolyl hydroxylase (EC 1.14.11.2) synthesis in chick embryo corneas. Increased cell density elevates enzyme production, regulated at the transcriptional level.
Area of Science:
- Biochemistry
- Cell Biology
- Developmental Biology
Background:
- Prolyl hydroxylase (EC 1.14.11.2) is crucial for collagen synthesis.
- Cellular microenvironment, including cell density, can influence enzyme production.
- Understanding enzyme regulation is key to cellular function.
Purpose of the Study:
- To investigate the effect of cell density on prolyl hydroxylase synthesis.
- To determine the regulatory mechanisms controlling prolyl hydroxylase production in cultured corneal cells.
Main Methods:
- Cultured corneas from 17-day-old chick embryos were used.
- Prolyl hydroxylase levels were quantified using immunoelectrophoresis.
- Synthesis rates were assessed using 35S-labeling; mRNA levels were measured with 32P-labeled cDNA.
Main Results:
- Prolyl hydroxylase amounts were dependent on cell density.
- High cell density (crowding) led to a two-fold increase in prolyl hydroxylase synthesis.
- A corresponding increase in prolyl hydroxylase mRNA levels was observed.
Conclusions:
- Cell density is a significant regulator of prolyl hydroxylase synthesis.
- The data suggest that prolyl hydroxylase synthesis is regulated at the transcriptional level.
- Findings provide insights into enzyme regulation in response to cellular environment.
Abstract:
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.
