Human placental GnRH-like factors: parallel displacement in GnRH immuno- and receptor-binding assays can be caused by degradation of radiolabelled GnRH tracers

Abstract

Human term placental cytosol fractions decreased the specific binding of gonadotrophin-releasing hormone (GnRH) isoform tracers to placental membranes (and to rat pituitary GnRH receptors and anti-GnRH antibodies) in a dose-dependent manner, and in parallel to GnRH standard curves. However, cytosol fractions had little or no effect on the binding of two GnRH superagonist tracers. The specificity of placental binding sites for a range of GnRH-like and unrelated peptides was shown to be similar with GnRH isoforms or GnRH agonists as binding ligands, suggesting that isoforms and agonists did not bind to different forms of the GnRH-receptor. Inclusion of a cocktail of protease inhibitors during the preparation of placental cytosol significantly reduced immuno- and receptor-binding activity. Moreover, incubation of radiolabelled chicken GnRH II with placental cytosol led to marked inactivation of tracer, as assessed by radioreceptor and radioimmunoassays for GnRH, high resolution liquid chromatography, thin layer chromatography and adsorption to dextran-coated charcoal and other matrices. There was a good negative correlation between tracer degradation and apparent GnRH immuno- and receptor-binding activities. These results emphasize the important effects which proteases in un-denatured tissue extracts can have on radioreceptor and radioimmunoassays due to inactivation of peptide tracers, and suggest that previous measurements of receptor- and immuno-active GnRH-like factors may have been over-estimated due to peptidase action during the GnRH assay.