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Measurement of Growth Hormone Synthesis by Rat Pituitary Cells in Culture^*

Department of Biological Sciences, Columbia University New York, New York 10027

Abstract

A direct immunoprecipitation assay for newly synthesized, radioactive rat growth hormone (GH) has been developed. Anti—GH plasma prepared in a baboon was employed to precipitate GH from the cytoplasm or growth medium of rat pituitary tumor cells (GH₃) exposed to ¹⁴C–amino acids in suspension culture. The identity of the precipitated radioactive material as GH was confirmed by acrylamide gel electrophoresis of the precipitate in the presence of sodium dodecyl sulfate, by interference with the immune precipitation of radioactivity by authentic GH, and by double immunodiffusion. The specificity of the assay for GH was demonstrated by gel electrophoresis of the precipitated material, and by the observation that normal baboon plasma precipitated only 5–15% as much radioactivity from labeled cytoplasm as did immune plasma. A 2% cross reaction of the anti–GH plasma with rat prolactin was observed. The precipitation of radioactive GH by the immune plasma was estimated to be 97% complete. Over a 10–fold range of concentration of radioactive GH, the radioactivity precipitated by the immune plasma was proportional to the amount of radioactive GH present. Using this technique, GH was found to represent 8% of the total protein synthesis by the GH3 cells. (Endocrinology 92: 1014, 1973

Footnotes

^* This work was supported by Grant GB–27412 from the National Science Foundation, and Grant BC–38 from the American Cancer Society.

Received August 1, 1972.