Incorporation of cis-hydroxyproline into protocollagen and collagen. Collagen containing cis-hydroxyproline in place of proline and trans-hydroxyproline is not extruded at a normal rate.

Incubation of cartilage from chick embryos with l*C3,5-cis-hydroxy-DL-proline demonstrated that cis-hydroxyproline is incorporated into the collagen and other protein synthesized by the tissue. Gel filtration of extracts from the tissue demonstrated that the proteins synthesized in the presence of cis-hydroxyproline were about the same size as proteins synthesized under control conditions. Incubation of the tissue with both SH-puromycin of known specific activity and r4C-cis-hydroxyproline made it possible to estimate the degree to which the analogue replaced proline and hydroxyproline in collagen. The results indicated that with 48 pg per ml of cis-hydroxyproline, about 10 % of the proline and hydroxyproline residues in collagen were replaced by the analogue. With 194 pg per ml of the analogue, about 61% of the proline and hydroxyproline residues were replaced by cis-hydroxyproline. Incubation of the tissue with 14C-proline or 14C-lysine and with cis-hydroxyproline indicated that the proline and lysine still incorporated into collagen in the presence of the analogue were hydroxylated and glycosylated to a normal extent. Autoradiographs of the tissue indicated that the collagen containing cis-hydroxyproline was not extruded into the extracellular matrix at a normal rate. The decreased rate of extrusion of collagen containing cishydroxyproline can be explained in one of two ways. (a) The presence of cis-hydroxyprolme in the collagen molecules specifically interferes with extrusion, or (b) the substitution of cis-hydroxyproline at sites in protocollagen which are normally converted to trans-hydroxyproline prevents the transhydroxyproline content from reaching a level necessary for extrusion from the cell at a normal rate.