SUMMARY
1. With rat diaphragm in vitro, addition of insulin to the medium so as to give a concentration as low as 0·05 mu./ml. of the hormone, stimulated the incorporation of [14C]glycine into protein of tissue. Simultaneous addition of glucose to the medium did not affect either the minimal amount of insulin required to produce a significant stimulation of incorporation of glycine, or the magnitude of the effect of the small concentration of insulin used.
2. Addition of a mixture of oxidized A and B chains of the insulin molecule did not affect incorporation of a mixture of labelled amino acids into the protein of isolated diaphragm, but a degraded insulin (DHA-insulin), which has about 15% of the activity of insulin in stimulating glucose uptake by diaphragm, was found to stimulate incorporation of [14C]glycine to an extent comparable with its effect in stimulating glucose-uptake.
3. Addition of rat serum, or the dipping of diaphragm in a medium containing insulin, stimulated incorporation of [14C]glycine into protein of diaphragm. Both these effects and the stimulation produced by insulin in vitro were abolished when the medium contained an antiserum to insulin.
4. Addition in vitro of growth hormone (GH) stimulated incorporation of [14C]glycine into protein of diaphragm from the hypophysectomized rat but had no effect on diaphragm from the normal rat, whether or not a small dose of insulin was also added in vitro. The action of GH in promoting incorporation of [14C]glycine into protein of diaphragm from the hypophysectomized rat was not neutralized by insulin antiserum.
5. Corticotrophin, cortisol, thyroxine, vitamin B12, vitamin D2 and linoleic acid all had no observable effect on incorporation of labelled amino acids into diaphragm. Glucagon stimulated incorporation, but the stimulation was abolished by the in vitro addition of antiserum to insulin and was probably attributable to the presence of a trace of insulin in the glucagon.
6. Anaerobiosis, and the addition of various metabolic inhibitors, were found to suppress incorporation of [14C]glycine into diaphragm protein almost entirely.
In vitro incorporation of nonnatural amino acids into protein using tRNACys-derived opal, ochre, and amber suppressor tRNAs
