In VitroBiosynthesis of Luteinizing Hormone in the Anterior Pituitary Gland1

Abstract

Isolated pituitary glands of rats, rabbits and sheep were incubated with C14-leucine in Krebs-Henseleit medium under an atmosphere of 95% O2 and 5% CO2 at 37 C. After incubation, the pituitary glands were homogenized, frozen, thawed and contrifuged at 2000Xg. A precipitate was obtained from the supernatant by adding rabbit anti-ovine LH-antiserum. The precipitate was further purified and its radioactivity was quantitatively measured. The incorporation of the radioactivity into the precipitate was time-dependent and was inhibited by puromycin. The radioactivity of the precipitate was markedly reduced when the anti-LH-antiserum had been previously absorbed with LH, indicating that the precipitable substances consisted mainly of LH itself or immunologically related substances. Castration and immunization of the animals with LH markedly increased the radioactivity incorporated into the precipitate. Hydrochloric acid digestion of the precipitate followed by a chromatographic study showed that the radioactivity of the LH and protein fractions was exclusively present in the leucine fraction. These facts suggest that de novo synthesis of LH in the anterior pituitary can be estimated in vitro from the radioactive amino acid incorporation into the substances which are precipitated by anti-LH-antiserum by the present procedure.