Modification of human placental lactogen with plasmin. Preparation and characterization of a modified hormone with increased biologic activity

Abstract

To determine the structure needed for the biologic activity of human placental lactogen (hPL), we have cleaved hPL with the proteolytic enzyme plasmin. Plasmin modified hPL (PL-hPL) was purified by gel chromatography. Analysis by sodium dodecyl sulfate-polyacrylamide gel electrophoresis before and after reduction showed that cleavage had occurred within the Cys53-Cys165 loop and tryptic peptide maps revealed that a single peptide consisting of residues 135 to 140 had been removed. 5-Dimethylaminonaphthalene-1-sulfonyl end group analysis and digestion with carboxypeptidase B confirmed that cleavage was complete and only the single hexapeptide was removed. In a membrane binding assay for lactogenic activity PL-hPL was 2- to 3-fold more potent than hPL. Using growth hormone receptors from rabbit liver membranes, PL-hPL was also more potent than hPL, but still much less potent than growth hormone. The lactogenic activity of PL-hPL in an in vitro bioassay was 75% above that of unmodified hormone. It is concluded that plasmin cleaves homologous peptides from hPL and growth hormone and that removal of the hexapeptide from hPL results in enhanced biologic activity.