Chinese hamster lung cell polysomes direct the synthesis of a single molecular weight species of procollagen alpha chains

Abstract

Polysomes prepared from cultured Chinese hamster lung cells direct the synthesis of procollagen alpha chains in an heterologous cell-free system containing the postribosomal supernatant fraction prepared from wheat germ. Total protein synthesis requires both subcellular components and an exogenous energy source, and is inhibited by the antibiotics puromycin and aurin tricarboxylic acid. The ratio of collagenase-digestible to nondigestible material produced depends upon the wheat germ and not the polysome level in the reaction. Under optimal conditions, a significant fraction of the total product migrates on denaturing sodium dodecyl sulfate-polyacrylamide gels as a single molecular weight collagenase-digestible species corresponding in size to the procollagen alpha chain (Mr approximately equal to 170,000). Approximately one-third of this high molecular weight material represents products whose synthesis results from cell-free mRNA initiation, and no distinct product larger than the 170,000-dalton material is observed. These studies confirm the initial observation that collagen represents one of the major gene products of Chinese hamster lung cells and demonstrate the usefulness of this cell line for the study of mammalian collagen biosynthesis.