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

Abstract

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.