In VitroEffects of Growth Hormone on the Number and Activity of Ribosomes Engaged in Protein Synthesis in the Isolated Rat Diaphragm1

Abstract

The following study was performed to determine if the in vitro stimulatory effect of growth hormone on protein synthesis in the rat diaphragm involves an increase in the number or activity of functional ribosomes in the cells. Isolated diaphragms of hypophysectomized rats were preincubated with bovine growth hormone at 37 C to activate the protein synthetic mechanism. Then they were incubated with ³Hpuromycin, which forms a peptide bond with nascent peptides on the functional ribosomes. Incubation with ³H-puromycin was carried out either in the presence of 1 mM emetine or at 2 C in order to prevent the formation of new nascent peptides during the course of the puromycin reaction. In the presence of emetine, the labeling of nascent peptides with the antibiotic proceeded in a curvilinear fashion and reached completion within 4–6 hr. The rate of labeling at 2 C was much slower. When diaphragms were preincubated for 1 hr with growth hormone and then incubated with ³H-puromycin in the presence of emetine for 6 hr to permit the labeling of nascent peptides to proceed to completion and thus give an assessment of the total pool of ribosomes engaged in protein synthesis, no effect of the hormone on the extent of labeling was detected. In contrast, when similarly treated diaphragms were incubated for only 1 hr with the tracer in the presence of emetine or for 3 hr at 2 C to determine the effect of the hormone on the rate of the reaction of puromycin with nascent peptides, a significant stimulatory effect of the hormone was observed. These findings suggest that the in vitro stimulatory effect of growth hormone on protein synthesis in the rat diaphragm is not due to an increase in the number of ribosomes engaged in protein synthesis. Rather, the effect appears to be related, at least in part, to an enhanced ability of the ribosome to promote peptide bond formation. The possibility that the hormone enhances ribosomal activity by affecting the peptidyl transferase reaction or the translocation process is discussed. (Endocrinology88: 1054, 1971)