Intracellular location of triple helix formation of collagen. Enzyme probe studies.
Primary cultures of chick embryo fibroblasts were used to study ribosomal events in the processing of procollagen. Polyribosomes from radiolabeled cells were subjected to enzyme probe analysis using collagenase and pepsin digestion to assess both the amount of procollagen present on the polyribosomes and the conformation of the molecule. The peptides rendered dialyzable by each enzyme treatment were analyzed for radioactive proline and hydroxyproline. Approximately 30% of the nascent proteins were collagenous. Although some hydroxyproline was dialyzable in the pepsin-treated material, a low ratio of hydroxyproline to proline (0.04) indicated that considerable amounts of noncollagenous proteins were digested. Polyribosomal material, previously treated with pepsin, was digested with purified collagenase. Similarly, collagenase-digested polyribosomes were treated with pepsin. The pepsin pretreatment released noncollagenous protein and served to purify the remaining ribosomally bound pepsin-resistant collagenous protein. Collagenase treatment of the pepsin-resistant ribosomally bound peptides released peptides with a hydroxyproline to proline ratio of 0.65, indicating that considerable hydroxylation of proline occurs on nascent ribosomally bound procollagen. This finding combined with the well documented stabilizing effect of hydroxyproline on the collagen triple helix and the demonstrated resistance of ribosomally bound procollagen to pepsin digestion indicates that the collagen triple helix may well form on the polyribosome.
D000818:Animals; D002458:Cell Fractionation; D002499:Centrifugation, Density Gradient; D002642:Chick Embryo; D003094:Collagen / Q000378:metabolism; D005347:Fibroblasts / Q000378:metabolism; D046911:Macromolecular Substances; D003012:Microbial Collagenase; D010434:Pepsin A; D011132:Polyribosomes / Q000378:metabolism; D011347:Procollagen / Q000378:metabolism; D011487:Protein Conformation; D012260:Ribonucleases
D046911:Macromolecular Substances; D011347:Procollagen; D003094:Collagen; D012260:Ribonucleases; D010434:Pepsin A; D003012:Microbial Collagenase