| Abstract: | Prolyl 4-hydroxylase (EC 1.14.11.2) is a key enzyme in collagen biosynthesis, its active form is a tetramer (alpha 2 beta 2). In L-929 fibroblasts in the log phase of culture there is a low level of active enzyme. When the cell culture reaches confluency, prolyl hydroxylase activity in cells increases by a process that requires de novo RNA and protein synthesis. The same result may be achieved by crowding the cells (replating log phase cells at the density of stationary phase cells). In the work reported here we further examined induction of the enzyme. RNA synthesis necessary for enzyme induction is complete 6 h after "crowding" while protein synthesis requires 12 h. Thymidine (0.2-0.5 mM) added to log phase cells will also cause enzyme induction to the level found in "crowded" or resting cells. We also looked at the decay of the enzyme activity after subculture. This occurs rapidly (enzyme half-life is 1-2 h) and is concurrent with the re-entry of resting cells into cell cycle; however, thymidine added at the time of subculture to block DNA synthesis does not prevent the loss of prolyl hydroxylase activity. These results suggest that when cells are not engaged in propagation, they begin to synthesize luxury proteins such as prolyl hydroxylase. However, the loss of prolyl hydroxylase during subculture is probably not a direct consequence of DNA synthesis. |
