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

R.A Maurer,D.J Mckean

doi:10.1016/s0021-9258(19)46930-5

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.

Highlights

These results strongly suggest the synthesis of preprolactin in intact pituitary cells
The present findings demonstrate that preprolactin is synthesized in pituitary cells and suggests that preprolactin is the biosynthetic precursor of prolactin
The demonstration of a cell-free system which synthesizes preprolactin and accurately cleaves it to prolactin is consistent with this view

Summary

Introduction

Following cell-free protein synthesis, reactions were diluted with an equal volume of NaCl/P,, 2% Triton X-100, 2% deoxycholate, 0.02 M leucine and centrifuged at 10,000 x g for 10 min. The cell-free product was immunoprecipitated in the presence of carrier prolactin and subjected to sequence analysis as described previously [10]. Analysis by polyacrylamide gel electrophoresis of products labeled by a 3-min pulse with [“Hlleucine demonstrated the synthesis of several peaks of immunoreactive radioactivity (Fig. 1A).

Results

Conclusion