Cell-free synthesis of a prolactin precursor directed by mRNA from cultured rat pituitary cells

Polyadenylate-rich mRNA extracted from an established line of functional rat pituitary tumor cells (GH3) which respond to the addition of thyrotropin-releasing factor with the synthesis and secretion of prolactin directed cell-free synthesis using a cell-free protein-synthesizing system derived from wheat embryos. The products of translation, when analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and fluorography, did not include any protein co-migrating with rat prolactin. One prominent protein synthesized was precipitated using anti-prolactin antiserum. This protein was larger than the prolactin standard with an apparent molecular weight of 28,000 +/- 2,000. Addition of a GH3 cell extract to the translation system resulted in the appearance of a protein which co-migrated with rat prolactin and was immunoprecipitated with anti-rat prolactin antiserum. Addition of thyrotropin-releasing factor to spinner cultures of GH3 cells for 48 to 72 hours increased de novo prolactin biosynthesis. Levels of mRNA coding for prolactin determined by translation of total mRNA and quantitation of preprolactin synthesized were stimulated 3- to 6-fold relative to the levels in control cultures, suggesting that regulation of prolactin synthesis was accomplished, at least in part, at a transcriptional level.