In order to determine the chemical features of human placental lactogen (hPL) necessary for its biologic activity we prepared the following fragments from the plasmin-cleaved hormone: reduced and alkylated 1-134, reduced and alkylated , and a 1-134 dimer joined through the single cysteinyl residue at position 53. In a radioimmunoassa using antibodies against native hPL, the two reduced and a?1 kylated fragments produced nonparallel displacement and had less than 1% of the activity of hPL. The ability of reduced and alkylated 1-134 to bind to mammary gland receptors was less than 5% of that of hPL; reduced and alkylated We have reported (1) that cleavage of human placental lactogen (hPL) with plasmin produces a modified form of the hormone (PL-hPL) missing hexapeptide 135-140 and having enhanced biologic activity compared to intact hPL. The cleaved hormone consisted of two fragments, residues 1-134 and residues 141-191, which remained covalently linked through the disulfide bond between cysteine-53 and cysteine-165. We have isolated the two fragments produced by plasmin digestion of hPL and characterized their biologic properties. The PL-hPL fragments were separated after reduction and alkylation or reduction without alkylation, but in the continuous presence of reducing agent. In the latter procedure the 1-134 fragment quantitatively formed a disulfide linked dimer, (1-134)2, upon removal of reducing agent. The immunologic and biologic properties of reduced and alkylated 1-134 (RCAM-1-134), reduced and alkylated , and (1-134)2 are the subjects of this report.
MATERIALS AND METHODShPL, kindly supplied by P. Bell and C. Bauer, was subjected to further purification over a column of Sephadex G-100 (5.0 X 95 cm) eluted with 0.05 ammonium bicarbonate. Plasmin digestion of hPL was carried out as described (1). PL-hPL was reduced with a 100-fold excess of 2-mercaptoethanol in 0.05 M NH4HCO3/8 M urea, pH 7.8, and alkylated with iodacetamide (twice recrystallized) according to the procedure of Crestfield et al. (2). Reduced and alkylated fragments were separated by gel filtration on a column of Sephadex G-100 (2.5 X 95 cm) eluted with 0.05 M NH4HCO3/8 M urea. When the fragments were not alkylated prior to separation, 0.01% dithioerythritol was added to the elution buffer. Fragments were desalted by chromatography on a column (2.5 X 45 cm) of Sephadex G-25 eluted with 0.05 NH4HCO3. The presence of free sulfhydryl groups in (1-134)2 or in the reduced and alkylated fragments was determined by reaction with Ellman's reagent (3). Sodium dodecyl sulfate/polyacrylamide gel electrophoresis was carried out according to a modification of the procedure of Weber et al. (4) in which indicated samples were not boiled in 2-mercaptoethanol prior to electrophoresis. Gels were stained with Coomassie blue. The radioimmunoassay of hPL was performed by a double antibody method (5, 6). Antibodies to purified hPL were produced in guinea pigs. The binding of hPL, RCAM-1-134, 2 to mammary gland membranes isolated from rabbits in midpr...