Effects of Incorporation of trans-4,5-Dehydrolysine on Collagen Biosynthesis and Extrusion in Embryonic Chick Tibiae

Abstract

Abstract Crystalline, chromatographically homogeneous dl-4,5-trans-dehydrolysine (dehydrolysine) was synthesized. The dehydrolysine specifically competitively inhibited the activation of [14C]lysine with transfer RNA and lysyl synthetase prepared from whole chick embryo. The dehydrolysine had little effect when tested with other amino acids. The Km for lysine was 21 µm and the Ki for dehydrolysine was 639 µm. When embryonic chick tibia were incubated with dehydrolysine for 5 to 22 hours they incorporated the dehydrolysine into protein at a linear rate. From the amino acid analysis of the tibiae incubated with 4.6 mm dehydrolysine it appeared that there was nearly complete replacement of lysine by dehydrolysine in the newly synthesized protein. This was confirmed by experiments in which tibiae were incubated with 14C-amino acids in the presence and absence of dehydrolysine. As the concentration of dehydrolysine in the incubation medium was increased there was a progressive decrease in the incorporation of [14C]lysine so that at 4.6 mm dehydrolysine there was 79% inhibition. A much smaller decrease in the incorporation of [14C]proline, [14C]glycine, [14C]alanine, or a mixture of 14C-amino acids was observed in the presence of dehydrolysine. Incubation of the cartilage with [14C]lysine or [14C]proline indicated that the lysine and proline still incorporated into collagen in the presence of dehydrolysine were hydroxylated to a normal extent. Thus collagen molecules containing a normal amount of trans-hydroxyproline but a greatly reduced amount of hydroxylysine and glycosylated hydroxylysine were synthesized. Autoradiographs indicated that the collagen containing dehydrolysine was not extruded into the extracellular matrix at a normal rate. It seems unlikely that this effect is due simply to the presence of dehydrolysine in the collagen since this analogue probably would not alter the collagen conformation. It appears therefore that a normal level of hydroxylysine is a necessary but not sufficient condition for normal extrusion.