The single tryptophan residue of human placental lactogen. Effects of modification and cleavage on biologic activity and protein conformation.
In order to define further the chemical features of the human placental lactogen (hPL) molecule responsible for its lactogenic activity, two derivatives of the hormone were prepared by treatment with BNPS-skatole (2-nitrophenylsulfenyl)-3-methyl-3'-bromoindolenine). At a molar ratio of reagent to hPL of 7:1, a derivative was produced in which the single tryptophan was completely oxidized. At higher ratios, a second derivative was formed in which the peptide chain was cleaved at the tryptophan residue and the two resulting fragments remained bound by the disulfide bond between Cys53 and Cys165. Oxidation of the single tryptophan resulted in reduced immunologic activity, reduced helical content as measured by circular dichroism below 240 nm, and changes in the near-UV circular dichroic spectrum, each indicating a change in the conformation of the hPL molecule. Nevertheless, this derivative retained 20% of its ability to bind to lactogenic receptors and 40 to 50% of its ability to stimulate N-acetyllactosamine synthetase in vitro. Cleavage at the tryptophan was not complete, but the loss of immunologic and biologic activity was equivalent to the degree of cleavage, indicating that the cleaved derivative was completely inactive. In addition, separation of the cleaved fragments from intact hormone followed by recombination did not generate any immunologic or biologic activity. We conclude that the single tryptophan of hPL is not essential for the biologic activity of hPL. It is likely that the reduced activity associated with modification or cleavage at the tryptophan residue is due to changes in the conformation of the molecule.
D000596:Amino Acids / Q000032:analysis; D000818:Animals; D001681:Biological Assay; D002942:Circular Dichroism; D005260:Female; D007700:Kinetics; D007774:Lactation; D008321:Mammary Glands, Animal / Q000378:metabolism; D010928:Placental Lactogen / Q000378:metabolism; D011247:Pregnancy; D011487:Protein Conformation; D011817:Rabbits; D011863:Radioimmunoassay; D011956:Receptors, Cell Surface / Q000378:metabolism; D018000:Receptors, Peptide; D012862:Skatole / Q000031:analogs & derivatives; D014364:Tryptophan
D000596:Amino Acids; D011956:Receptors, Cell Surface; D018000:Receptors, Peptide; C028202:placental lactogen receptor; C005904:BNPS-skatole; D014364:Tryptophan; D010928:Placental Lactogen; D012862:Skatole