Hydroxylation of prolyl residues in type II procollagen in vitro and in cellulo. Lack of preferential hydroxylation of specific regions of the protein.
[14C]Proline-labeled protocollagen, the unhydroxylated form of procollagen, was isolated from cartilage cells incubated with alpha, alpha'-dipyridyl. For examination of the initial steps in the hydroxylation of the protein, it was incubated in vitro with prolyl hydroxylase so that an average of 1.3-2.7 prolyl residues per chain was hydroxylated. The partially hydroxylated alpha chain were cleaved with cyanogen bromide, and the fragments were separated by polyacrylamide gel electrophoresis or column chromatography. The cyanogen bromide fragments were hydroxylated to the same degree. The results indicated, therefore, that in the initial hydroxylation of alpha chains in vitro, there was no preferential hydroxylation of any specific regions of the protein. In a second series of experiments, cartilage cells were incubated with [14C]proline and alpha, alpha'-dipyridyl so that prolyl hydroxylase in the cells was extensively, but not completely, inhibited. Partially hydroxylated alpha chains were isolated, and cyanogen bromide fragments of the alpha chains from the cells were assayed for hydroxy[14C]proline. The alpha chains contained an average of two residues of hydroxyproline per chain, and the cyanogen bromide fragments were hydroxylated to about the same degree. The results indicated, therefore, that when prolyl hydroxylase activity in cells is low relative to the rate at which pro alpha chains are synthesized, hydroxylation of prolyl residues occurs as it does in vitro, and there is no preferential hydroxylation of a specific region of the protein.
D000818:Animals; D002356:Cartilage / Q000378:metabolism; D002478:Cells, Cultured; D002642:Chick Embryo; D002845:Chromatography; D004591:Electrophoresis, Polyacrylamide Gel; D006900:Hydroxylation; D006909:Hydroxyproline / Q000378:metabolism; D010446:Peptide Fragments / Q000378:metabolism; D011347:Procollagen / Q000378:metabolism*; D011393:Procollagen-Proline Dioxygenase / Q000378:metabolism; D011392:Proline / Q000378:metabolism*
D010446:Peptide Fragments; D011347:Procollagen; D011392:Proline; D011393:Procollagen-Proline Dioxygenase; D006909:Hydroxyproline