BIOSYNTHESIS OF HUMAN CHORIONIC GONADOTROPHIN IN VITRO: INCORPORATION OF [14C]l-LEUCINE

Abstract

SUMMARY Fresh human placental tissue from first trimester pregnancies was incubated with [14C]l-leucine and the incorporation of the labelled amino acid into placental proteins was studied in both the soluble and the particulate fractions. Free [14C]l-leucine was completely separated from labelled proteins by exhaustive dialysis. This was confirmed by Sephadex G-100 gel chromatography. In preliminary experiments, the total protein fraction from Sephadex was submitted to polyacrylamide gel electrophoresis; columns divided into ten sections were checked, fraction by fraction, for incorporated radioactivity and human chorionic gonadotrophin (HCG) potency, using a radioimmunoassay method. In all instances, the highest HCG activity corresponded to the highest incorporation of radioactivity. In subsequent experiments only those radioactive proteins were used which had the highest activity after chromatography. This peak was further divided into two parts which were submitted to polyacrylamide gel electrophoresis. Using the ascending limb of the peak which contained about 70% of the total HCG activity, there was complete agreement between the distribution of radioactivity and that of HCG activity, whereas the bulk of HCG radioimmunological activity in the descending limb could be dissociated from radioactive proteins. After absorption of the HCG-like protein with specific anti-HCG serum, HCG activity was completely neutralized and more than 95% of the corresponding radioactivity disappeared. Additional evidence for the presence of an HCG-like biosynthesized protein was obtained after DEAE-cellulose ion exchange chromatography of the products of the incubation. Here again, the HCG potency of the radioactive protein with the highest activity was completely neutralized after absorption with a specific antiserum: in this case, however, only some 60% of the total radioactivity present disappeared after absorption. The results indicate that human placental tissue can synthesize from l-leucine proteins possessing radioimmunological HCG activity.