Synthesis and Secretion of Hydroxyproline-containing Macromolecules in Carrots: II. In vivo Conversion of Peptidyl Proline to Peptidyl Hydroxyproline

Abstract

The chelator alpha,alpha'-dipyridyl prevents the formation of protein-bound hydroxyproline without affecting protein synthesis as measured by the incorporation of (14)C-proline. The inhibitory effect can be overcome by exogenous ferrous ions. Neither alpha,alpha'-dipyridyl nor Fe(2+) interferes in any other known way with the synthesis and secretion of the hydroxyproline-rich proteins normally found in the cell wall. This reversal by Fe(2+) of the inhibition of proline hydroxylation by alpha,alpha'-dipyridyl is used for a study in vivo of the hydroxylation reaction. This reaction has a temperature coefficient of 2.2, suggesting that it is an enzymatic process. Reversal by Fe(2+) of the alpha,alpha'-dipyridyl inhibition can occur after protein synthesis has been arrested with cycloheximide, indicating that peptidyl proline is the substrate of the hydroxylation reaction. Hydroxylation occurs in the cytoplasm, and not in the cell wall, and only for a limited time after the incorporation of proline into the polypeptide chain. This suggests a spatial separation of the enzyme and the substrate.