Biochemical characterization of collagens and of a non-collagenous protein synthesized by guinea pig lung fibroblasts in culture.
Employing various radioactive amino acids, protein biosynthesis by guinea pig lung fibroblasts has been studied in monolayer culture. The cells were shown to synthesize and secrete several collagenous and noncollagenous proteins. The biosynthesized macromolecules have been characterized employing molecular sieve and ion exchange chromatography (DEAE-cellulose column), SDS-polyacrylamide gel electrophoresis, and enzymatic digestion. It was found that the guinea pig lung fibroblasts synthesized mainly type I procollagen molecules which appeared in the media in various stages of cleavage. The intact procollagen molecules and the various processed products were identified by their electrophoretic migration in SDS-polyacrylamide slab gels and further characterized by 1) elution position on SDS-agarose columns under reducing conditions; 2) hydroxyproline content; 3) change in elution position on SDS-agarose after pepsin digestion; 4) chromatographic separation on DEAE-cellulose columns and electrophoretic mobility of the various peaks; and 5) susceptibility to collagenase digestion. Slab gel electrophoresis under non-reducing conditions of aliquots of culture media after limited pepsin digestion indicated the presence of disulfide-bonded type III collagen alpha-chains. In addition, the lung fibroblasts synthesized a non-collagenous protein composed of two disulfide-bonded chains. The individual chains appeared to have a molecular weight of approximately 220,000 when examined under reducing conditions. This protein has been identified as fibronectin based on its molecular weight, its resistance to collagenase attack, its susceptibility to protease digestion and its precipitation with specific antisera to fibronectin.
D000818:Animals; D002478:Cells, Cultured; D011232:Chemical Precipitation; D002848:Chromatography, DEAE-Cellulose; D002850:Chromatography, Gel; D003094:Collagen / Q000096:biosynthesis*; D004591:Electrophoresis, Polyacrylamide Gel; D005347:Fibroblasts / Q000378:metabolism; D005353:Fibronectins / Q000096:biosynthesis*; D006168:Guinea Pigs; D006909:Hydroxyproline / Q000096:biosynthesis; D007120:Immunochemistry; D008168:Lung / Q000378:metabolism*; D011347:Procollagen / Q000096:biosynthesis; D011392:Proline / Q000378:metabolism
D005353:Fibronectins; D011347:Procollagen; D003094:Collagen; D011392:Proline; D006909:Hydroxyproline