Biochemical characterization of procollagen-collagen synthesized by rabbit corneal endothelial cells in culture.
Collagen synthesis was studied in monolayer cultures of rabbit corneal endothelial cells by following [14C]proline and [3H]glucosamine or [3H]fucose incorporation into a fraction enriched for collagen and its precursor molecules. Sodium dodecyl sulfate gel electrophoresis of this fraction showed that it consisted of a high-molecular-weight (greater than 300,000 daltons) polypeptide. This component was collagenase sensitive and, in the presence of dithiothreitol, gave rise to two polypeptides of the apparent molecular weights of 200,000 and 160,000 daltons. Pepsin digestion of this material destroyed all the high-molecular-weight material and gave rise to a single collagenase-sensitive component of an apparent molecular weight of 115,000 daltons. This 115,000 dalton material is similar to previously observed basement membrane collagens, and the 160,000 and 200,000 dalton components are probably precursor chains of basement membrane collagen. The very-high-molecular-weight material (greater than 300,000 daltons) may represent a disulfide-linked complex of these precursor chains. DEAE-cellulose column chromatography confirmed the presence of a single procollagen species distinct from the collagen fraction. Amino acid analysis of collagen and procollagen fractions showed a decreased hydroxyproline value as compared with previously reported basement membrane collagens or collagen precursors.
D000596:Amino Acids / Q000032:analysis; D000818:Animals; D002478:Cells, Cultured; D002848:Chromatography, DEAE-Cellulose; D003094:Collagen / Q000096:biosynthesis*; D003315:Cornea / Q000032:analysis / Q000378:metabolism* / Q000502:physiology; D003886:Descemet Membrane / Q000032:analysis; D004727:Endothelium / Q000032:analysis; D066298:In Vitro Techniques; D010455:Peptides / Q000032:analysis; D011347:Procollagen / Q000096:biosynthesis*; D011817:Rabbits
D000596:Amino Acids; D010455:Peptides; D011347:Procollagen; D003094:Collagen