Cell-free synthesis of putative type V procollagen chains programmed by Chinese hamster lung cell mRNA

A messenger RNA fraction isolated from cultured Chinese hamster lung (CHL) cells programs in a cell-free system prepared from wheat germ the efficient incorporation of [14C] proline into newly synthesized protein with a significant fraction of the incorporated substrate being digestible with bacterial collagenase. This reaction requires both subcellular fractions, an energy source, and is inhibited by the antibiotic puromycin. The relative amount of collagenase-digestible to non-digestible cell-free product depends upon the ratio of CHL mRNA to wheat germ lysate, is not affected by either the Mg2+ or K+ concentrations employed, and under optimal condition, approximately 38% of the total incorporated substrate is collagenase-sensitive. Electrophoresis on SDS-polyacrylamide gels of the products programmed by CHL mRNA indicates that the collagenase-digestible material corresponds in size to a procollagen chain with an apparent molecular mass of approximately 170,000 daltons. These studies suggest that the collagen alpha 1 (V) chain is initially synthesized as a precursor procollagen chain and demonstrate that a significant amount of the mRNA in Chinese hamster lung cells codes for this protein.