A study of the biosynthesis of growth hormone and prolactin in bovine pituitary slices and cell-free systems

Abstract

The biosynthesis of growth hormone and prolactin was studied in both slice and cell-free systems from bovine anterior pituitary tissue. Slice preparations were highly active in incorporating radioactive amino acids into prolactin t growth hormone and protein. Modification of existing procedures were employed for the isolation of the labeled hormones in a relatively purified state. A number of physical t chemical, and biological criteria. were used to demonstrate that the radioactive proteins were indistinguishable from reference standards. These included amino acid composition, N- and C-terminal analysis, gel filtration, ion-exchange chromatography, polyacrylamide gel electrophoresis, and Ouchterlony immunodiffusion. Using the same procedure, the relationship of growth hormone releasing factor (GRF) to the synthesis and release of growth hormone in vitro with both bovine and rat pituitaries was studied. Although no effect on synthesis and only a slight effect on release were noted, these results must be considered inconclusive because of the small number of samples studied. With a cell-free system, consisting of ribosomes plus pH 5 enzyme fraction, and fortified with ATP, GTP, and Mg++, the optimal conditions for biosynthesis of growth hormone and general protein were established. When a polysome-enriched preparation was resolved into fractions of discrete particle size on sucrose density gradients, incorporation of radioactive amino acids into the hormone was most active with polysomes containing six to seven ribosomal units. The isolated radioactive product was indistinguishable from the authentic growth hormone standard by four physical criteria; ion-exchange chromatography, Sephadex gel filtration, polyacrylamide disc gel electrophoresis and sucrose density gradient centrifugation. In addition, it displayed specific antibody binding. Examination by similar physical criteria, of the radioactive polypeptide associated with the prolactin fraction isolated from the pituitary ribosomal system, revealed no evidence of incorporation of labeled amino acids into the monomer form of prolactin. Several explanations have been offered for the failure to demonstrate synthesis of prolactin with this cell-free system.