mRNA dependent synthesis of authentic precursor to human placental lactogen: conversion to its mature hormone form in ascites cell free extracts

Messenger RNA derived from term placenta directs the synthesis of human placental lactogen (hPL, molecular weight 22,200) in an ascites 30,000 x g post mitochondrial supernate (S 30). When the S 30 is fractionated into ribosome and cell sap (S 100) fractions, and these are recombined for incubation, term placental mRNA directs the synthesis of a protein with a molecular weight of 25,000. This protein contains authentic hPL tryptic peptides. This suggested that during the separation of ribosomes and S 100 a component responsible for cleavage was lost. A 1.0 M sucrose cushion was used for the preparation of ribosomes and S 100 and membranous material accumulated at the sucrose interphase. When this membrane fraction was added back to the ribosome S 100 system only hPL was formed. Cleavage was greatest when membranes were added within the first few minutes of incubation. In a run off system composed of term polysomes, ascites S 100, and the inhibitor of initiation, pactamycin, the 25,000 molecular weight material, referred to as pre hPL, was also synthesized. These data strongly suggest that (1) pre hPL is an authentic precursor to hPL, (2) cleavage of the precursor primarily occurs on nascent, ribosome bound peptide chains, and (3) pre hPL is the primary gene product.