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 (i) pre-hPL is an authentic percursor to hPL, (ii) cleavage of the precursor primarily occurs on nascent, ribosome-bound peptide chains, and (iii) pre-hPL is the primary gene product.