The degredation of newly synthesized collagen in cell culture

PLEASE NOTE: This work is protected by copyright. Downloading is restricted to the BU community: please click Download and log in with a valid BU account to access. If you are the author of this work and would like to make it publicly available, please contact open-help@bu.edu.Thesis (M.Sc.D.)--Boston University, Henry M. Goldman School of Graduate Dentistry, (Oral Biology), 1982.Bibliography: leaves 60-65.The ability of ce11s in culture to degrade newly synthesized collagen has been demonstrated. This study was designed to characterize the products of this degradative process and if possible to determine the site of degradation. Fibroblast and smooth muscle ce= cultures were pulsed for 24 hrs with 14c-PrOline in medium supplemented with ascorbate and dialyzed serum. After filtration (25,000 daltons or greater exclu- Sion) the resulting filtrates were analyzed for the presence of free and/Or PePtide bound 14c-hydroxyproline. Assuming the total filterable hydroxyproline represented degraded co11agen, the fibroblast’cultures degrade 15-20% of the collagen synthesized during the pulse period while the muscle ce11s yield 10% degradation. Gel filtration on a Bio-Gel P-2 COlumn resulted in recovery of at least 90% of this filterable radioactive hydroxyproline as the free amino acid. The addition of cycIoheximide or α,α一〇dipyridyl to the medium during the pulse period se- Verely diminished the formation of the free hydroxyproline suggesting that protein synthesis and prolyl hydroxylase activity were required before the free hydroxyproline could be fomed. In summary we have identified free hydroxyproline as a major degradation product of newly Synthesized collagen in cell cultures. The hydroxyproline is of enzymatic and protein (collagen) origin and not due to nonspecific oxidation (or hydroxylation) of proline. These data and those obtained from ce11 Culture studies with leupeptin suggest that lysosomal enzymes are involved in the process of intracellular collagen degradation