Formation of collagen hydroxyproline in vitro

Abstract

When slices of granuloma induced by carrageenin were incubated in the presence of L-C14 -proline, both proline and hydroxyproline isolated from the soluble and insoluble collagen fractions were labeled. After a lag period of 5 minutes the specific activities of proline and hydroxyproline from neutral-salt-soluble collagen increased linearly with time for at least 45 minutes. After 4 hours the total counts in this fraction were 20 times those in the acid-soluble or insoluble collagen fractions, thus confirming neutral-salt-soluble collagen as an early stage in collagen-fiber formation. The specific activity of hydroxproline from neutral -salt-soluble collagen increased 0.6 to 1.6 times that of the proline during incubation for 4 hours. Addition of unlabeled L-hydroxyproline to the incubation medium in the presence of L-C14-proline had no effect on the ratio of the specific activities of collagen hydroxyproline and proline, but the total radioactivity incorporated was reduced. Incubation with L-C14-hydroxyproline did not produce a significant incorporation of radioactivity into collagen. Radioactive free hydroxyproline was isolated from the slices and medium after incubation with L-C14-proline, but its specific activity was only half that of the neutral-salt-soluble collagen hydroxyproline and the total counts present were not increased when unlabeled hydroxyproline was present as a trapping agent. Free hydroxyproline is not an intermediate in the formation of the hydroxyproline of collagen. Both proline and hydroxyproline added to the medium were concentrated intracellularly about 2.5 times. Lack of incorporation of free hydroxyproline cannot therefore be due to the impermeability of the cells towards hydroxyproline. The specific activity of the free proline from the slices increased to 20% of that in the incubation medium within 10 minutes but no further increase occurred in spite of the high specific activity of the proline from the medium. The latter fell to approximately one-third of its initial value during incubation for 4 hours, through dilution by proline liberated from the slices, but it was never less than twice the specific activity of the slice proline.