Collagen turnover and the growth state in 3T6 fibroblast cultures

The metabolic turnover of collagen in 3T6 fibroblast cultures was studied at various stages of growth by compartmental analysis of hydroxyproline content and of the conversion of radioactive proline to hydroxyproline. Quantitative and radioactive hydroxyproline measurements were directly parallel, although the latter method was more sensitive in demonstrating collagen synthesis in all phases of growth. Hydroxyproline formation as an index of collagen synthesis was abruptly accelerated at the time of confluence, and was well maintained at the new rate despite a gradual decline in the rate of general protein synthesis. Ethanol fractionation of growth medium proved superior to perchloric acid extraction in providing a pool of soluble hydroxyproline clearly derived from collagen degradation. Breakdown products were released at a constant rate per cell in all growth phases and accumulated to high levels in continuous culture. The fractional rate of collagen breakdown (percentage of hydroxyproline radioactivity) was highest in early logarithmic growth. Its gradual decline as cultures aged reflected the increased hydroxyproline content and improved stability of collagen under these conditions.