Effect of cycloheximide on collagen biosynthesis as evidence for a post-ribosomal site for the hydroxylation of proline.
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abstract
The site of the hydroxylation of proline during collagen biosynthesis was studied by using cycloheximide to prevent the release of newly-synthesized polypeptides from ribosomal complexes. When [14C]proline and cycloheximide were added simultaneously to isolated cartilage from chick embryos, only about 1 % of the 14C incorporated into non-dialyzable polypeptides was converted to [14C]hydroxyproline. When the [14C]proline which was incorporated in the presence of cycloheximide was chased with multiple changes of fresh media containing [12C]proline and no cycloheximide, about 20 % of the incorporated [14C]proline was converted to [14C]hydroxyproline. Control experiments indicated that cycloheximide did not directly inhibit the hydroxylation of proline in protocollagen within the tissue. The question of whether peptides bound to ribosomes can be hydroxylated was examined further with purified protocollagen hydroxylase, the enzyme which hydroxylates proline in protocollagen and synthetic polypeptides similar to protocollagen. Protocollagen hydroxylase in vitro did not hydroxylate the non-dialyzable [14C]proline in 15000 g supernatants of homogenates of tibiae which had been incubated with [14C]proline in the presence of cycloheximide. By contrast, when the labeled polypeptides in these homogenates were extracted from ribosomes with sodium dodecylsulfate or by boiling in urea, the protein-bound [14C]proline was hydroxylated by the enzyme. 1967.
