The Effects of Ascorbic Acid Deficiency on Collagen Synthesis by Mouse Molar Tooth Germs in Organ Culture

Second molar tooth germs from 2-day-old Swiss-Webster mice, grown in organ culture for 7 days in ascorbic-acid-deficient medium, synthesized about 65% as much protein (measured by incorporation of [14C]proline during a 24-h pulse) as did ascorbic-acid-supplemented controls. The newly synthesized proteins from ascorbic-acid-deficient cultures contained only about 7% of the hydroxyproline content of the controls. Collagenase digestion of the newly synthesized proteins showed that collagen comprised the same fraction of the total protein synthesized under both culture conditions. This result indicates that the ascorbate-deficient cultures made significant quantities of underhydroxylated collagen. Partial characterization of the collagen alpha chains on carboxymethyl cellulose columns showed an alpha1/alpha2 ratio of about 5, suggesting that at least two different species of collagen were synthesized. The alpha1/alpha2 ratio of the chains recovered from the ascorbate-deficient cultures was also about 5 but the chains were slightly underhydroxylated and the total amount of these chains which could be identified accounted for only a small fraction of the total collagen which was synthesized. A large fraction of the synthesized collagenous protein was found in the culture medium, mostly in the form of lower molecular weight peptides. It is concluded that most of the collagen which is synthesized by ascorbate-deficient tooth-bud cultures is not utilized by the component tissues, but is probably degraded and released into the medium.