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

Human term placental cytosol fractions decreased the specific binding of go nadotrophin-releasing hormone (GnRH) isoform tracers to placental membranes (and to rat pituitary GnRH receptors and anti-GnRH antibodies) in a dose-d ependent manner, and in parallel to GnRH standard curves. However, cytosol fractions had little or no effect on the binding of two GnRH superagonist t racers. 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 pr otease inhibitors during the preparation of placental cytosol significantly reduced immuno- and receptor-binding activity, Moreover, incubation of rad iolabelled chicken GnRH II with placental cytosol led to marked inactivatio n of tracer, as assessed by radioreceptor and radioimmunoassays for GnRH, h igh resolution liquid chromatography, thin layer chromatography and adsorpt ion to dextran-coated charcoal and other matrices. There was a good negativ e correlation between tracer degradation and apparent GnRH immuno- and rece ptor-binding activities. These results emphasize the important effects whic h proteases in un-denatured tissue extracts can have on radioreceptor and r adioimmunoassays due to inactivation of peptide tracers, and suggest that p revious measurements of receptor- and immune-active GnRH-like factors may h ave been over-estimated due to peptidase action during the GnRH assay.