Regulation of the glycosylations of collagen hydroxylysine in chick embryo tendon and cartilage cells.
The regulation of the glycosylations of hydroxylysine was studied in isolated chick-embryo cells by labelling with a [14C]lysine pulse. The course of the procollagen lysyl modifications was compared in tendon and cartilage cells, and the effect on the gycosylations of the degree of lysyl hydroxylation and the concentration of Mn2+ and Fe2+ were also studied, in tendon cells. Procollagen triple helix formation was inhibited in most experiments in order to eliminate the effect of this process on the continuation of the reactions. Both in the tendon and cartilage cells the intracellular lysyl modifications proceeded in a biphasic fashion. After an initial sharp linear increase, the reactions did not cease but were protracted at a slower but constant rate. Lysyl hydroxylation was followed by rapid galactosylation in both cell types and this was followed almost immediately by rapid glucosylation, suggesting a close association of the corresponding enzymes. The data further suggest that other factors must also exist, in addition to the differences in the timing of triple helix formation and the actual hydroxylysine content, which are responsible for the different amounts of galactose in the collagens synthesized by these cell types. The amount of glucosylgalactosylhydroxylysine nevertheless seemed to be determined by the available acceptor sites, i.e., the amount of galactosylhydroxylysine. In further experiments with tendon cells the oxygen participating in lysyl hydroxylation was displaced by nitrogen at various points in time. When the degree of lysyl hydroxylation was reduced to less than one-third of the original, the total amounts of glycosylated residues decreased correspondingly, but their proportion relative to total hydroxylysine remained unchanged. Extra Mn2+ increased the proportion of galactosylated hydroxylysine, suggesting that the activity of hydrosylysyl galactosyltransferase is not saturating in respect of the catalyzed reaction. Experiments on the addition of Fe2+ or its chelation by alpha, alpha'-dipyridyl gave indications that the presence of this co-factor is not required for either glycosylation reaction in isolated tendon cells.
D000818:Animals; D002356:Cartilage / Q000378:metabolism*; D002478:Cells, Cultured; D002642:Chick Embryo; D003094:Collagen / Q000096:biosynthesis*; D005690:Galactose / Q000378:metabolism; D005947:Glucose / Q000378:metabolism; D006900:Hydroxylation; D006901:Hydroxylysine / Q000378:metabolism*; D007501:Iron / Q000494:pharmacology; D008345:Manganese / Q000494:pharmacology; D011347:Procollagen / Q000378:metabolism; D013710:Tendons / Q000378:metabolism*
D011347:Procollagen; D006901:Hydroxylysine; D008345:Manganese; D003094:Collagen; D007501:Iron; D005947:Glucose; D005690:Galactose