Time course of release into the medium of newly synthesized proteins by cultured aortic endothelial cells. Role of serum in preventing proteolytic degradation.

Abstract

Endothelial cells from pig aortas were labeled with 35S-methionine, and the soluble proteins that were released into the culture medium were examined by SDS-PAGE. Proteins were collected during labeling, and during the intervals 0 to 3, 3 to 6, 6 to 12, and 12 to 24 hours of chase after a 1-hour labeling period, and during the intervals 0 to 12, 12 to 24, and 24 to 48 hours of chase after a 24-hour labeling period. Release of radiolabeled soluble protein from cells into the medium continued over the longest time periods examined. If the cells were labeled for 1 hour, characteristic patterns of proteins appeared in the medium during the labeling period, early (0- to 3-hour postlabeling) and late (6- to 24-hour postlabeling). The time course of release of fibronectin (Fn) and of angiotensin-converting enzyme (ACE) was determined by using immunoprecipitation. ACE appeared only after 6 hours of chase. Fn appeared throughout the chase period. The effects of serum and of the protease inhibitors alpha-2 macroglobulin, pepstatin, and leupeptin on protein release were examined. In the absence of serum, endothelial cell culture medium contained substantial protease activity capable of completely degrading most of the released proteins; a major effect of serum was to protect newly released protein from degradation.