The wheat germ cell-free system possesses processing activity for the precursor of human placental lactogen

Abstract

1. Total RNA was extracted from human term placenta and mRNA purified by chromatography on oligo(dT)-cellulose. The poly(A)-containing fraction stimulated amino acid incorporation 5- to 10-fold in the wheat germ cell-free system. Immunoprecipitation with an anti-lactogen serum indicated that 14-27% of the peptides synthesized in vitro contained antigenic determinants of this hormone. 2. Analysis of the [3H]leucine labelled product in the immunoprecipitate on sodium dodecyl sulfate-polyacrylamide gels revealed a complex mixture of polypeptides. Two heavily labelled bands (I and III) were seen corresponding in mobility with pre-lactogen (Mr = 25 000) and native lactogen (Mr = 22 200), each accounting for about 30% of the immunoprecipitable radioactivity. Two additional bands with an intermediate mobility were also observed. 3. Synthesis of the hormone was inhibited by 7-methylguanosine-5'-monophosphate suggesting the presence of a 7-methylguanosine 'cap' on the 5'-end of the mRNA for lactogen. 4. Peptide analysis of the cyanogen bromide cleavage products of band I, band III and authentic lactogen showed marked similarities in their primary structure. The precursor molecule, however, was lacking the N-terminal peptide present in authentic hormone indicating the presence of an extension of 25 amino acids at this side of the molecule. 5. The presence of one or several processing enzymes in the wheat germ cell-free system was indicated by the effect of Triton X-100. Low concentrations of this detergent (0.04%) while inhibiting the protein synthesizing activity for only 15%, completely abolished the precursor cleavage activity. Under these conditions only pre-lactogen was detected in the immunoprecipitate.