Collagen biosynthesis. Characterization of subcellular fractions from embyonic chick fibroblasts and the intracellular localization of protocollagen prolyl and protocollagen lysyl hydroxylases.
1. Subcellular fractions of freshly isolated matrix-free embryonic chick tendon and sternal cartilage cells have been characterized by chemical analysis, electron microscopy and the location of specific marker enzymes. These data indicate the fractions to be of a high degree of purity comparable with those obtained from other tissues, e.g. liver and kidney. 2. When homogenates were assayed for protocollagen prolyl hydroxylase and protocollagen lysyl hydroxylase activities, addition of Triton X-100 (0.1%, w/v) was found to stimulate enzyme activities by up to 60% suggesting that the enzymes were probably membrane-bound. 3. Assay of subcellular fractions obtained by differential centrifugation for protocollagen prolyl hydroxylase activity indicated the specific activity to be highest in the microsomal fraction. Similar results were obtained for protocollagen lysyl hydroxylase activity. 4. Submicrosomal fractions obtained by discontinuous sucrose-gradient centrifugation were assayed for the two enzymes and protocollagen prolyl hydroxylase and protocollagen lysyl hydroxylase were found to be associated almost exclusively with the rough endoplasmic reticulum fraction in both tendon and cartilage cells.
D000818:Animals; D002499:Centrifugation, Density Gradient; D002642:Chick Embryo; D003094:Collagen / Q000096:biosynthesis*; D004721:Endoplasmic Reticulum; D004789:Enzyme Activation; D005347:Fibroblasts / Q000032:analysis / Q000201:enzymology* / Q000648:ultrastructure; D008239:Lysine; D008566:Membranes; D008854:Microscopy, Electron; D006899:Mixed Function Oxygenases / Q000032:analysis*; D011092:Polyethylene Glycols / Q000494:pharmacology; D011393:Procollagen-Proline Dioxygenase / Q000032:analysis*; D013347:Subcellular Fractions; D013710:Tendons / Q000166:cytology
D011092:Polyethylene Glycols; D003094:Collagen; D006899:Mixed Function Oxygenases; D011393:Procollagen-Proline Dioxygenase; D008239:Lysine