Preparation of a disulfide-linked dimer of human placental lactogen fragment 1--134 with immunologic and biologic activity.

Abstract

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...