Biosynthesis of collagenase by human skin fibroblasts in monolayer culture.

The biosynthesis of human skin collagenase was studied in fibroblast cultures incubated with 13H]leutine under serum-free conditions. The radioactively labeled enzyme was isolated and quantitated directly from culture medium using a highly specific immunoprecipitation method. Sodium dodecyl sulfate gel electrophoresis of the immunoprecipitates showed two radioactive bands tihich co-migrated with the two proenzyme forms of purified human skin collagenase. F’urthermore, the immunoreactive enzyme protein coeluted with collagenase activity in ion exchange chromatography. The qualitative nature of the intracellular 3Hlabeled enzyme protein was examined by precipitation from cell lysates after partial purification. The results indicated that some of the intracellular enzyme protein exists in a form electrophoretically identical to the two proenzyme species of collagenase found in the culture medium. The labeled human skin collagenase represented 3 to 6% of the trichloroacetic acid-precipitable protein in the culture medium and 0.2 to 1.0% of the total newly synthesized 3H-labeled protein (medium + cells) produced under these serum-free conditions. Intracellular labeled enzyme protein could be detected after a 15min labeling period and reached a constant level at 45 to 60 min, after which there was no substantial increase in the intracellular storage of collagenase for up to 20 h. Secretion was linear once the intracellular labeled enzyme reached a constant level, a finding which suggests that the labeled enzyme had become equilibrated with the pre-existing intracellular enzyme pool and that the rate of secretion of the extracellular enzyme was largely reflective of de nouo synthesis.