Synthesis of preprolactin and conversion to prolactin in intact cells and a cell-free system

Monolayer cultures of pituitary cells were pulse-labeled with [³H]leucine for several minutes and the incorporated radioactivity was analyzed by immunoprecipitation and electrophoresis on sodium dodecyl sulfate containing polyacrylamide gels. Following a 3-min labeling period, a peak of radioactivity with a mobility similar to that of preprolactin was observed, as well as radioactivity co-migrating with prolactin. Competition with unlabeled prolactin demonstrated the specificity of the immunoprecipitation reaction. After 5 min of pulse-labeling followed by 5-min chase in medium with unlabeled leucine, only a product with the mobility of prolactin remained. Addition of a membrane fraction from dog pancreas to a wheat germ cell-free translation system containing pituitary mRNA resulted in the conversion of preprolactin to prolactin. Partial sequence analysis demonstrated that the processed product contained the correct NH₂ terminus of prolactin. Thus, both intact pituitary cells and a cell-free heterologous system are able to synthesize preprolactin and cleave it to prolactin offering strong evidence that preprolactin is the biosynthetic precursor to prolactin.