Human placental gonadotrophin-releasing hormone-like factors: an artefact of human placental peptidases?
Non-denatured human placental cytosol fractions displaced tracer binding in parallel with gonadotrophin-releasing hormone (GnRH) isoform and agonist peptides in GnRH-specific radioimmunoassays and radioreceptor assays. However, placental immuno- and receptor binding-GnRH-like activity was highly correlated with inactivation of GnRH tracers, suggesting that placental GnRH-like factors may be an artefact of ligand degradation during assay. The properties and inhibitor sensitivities of the major (125)I-labelled GnRH-degrading enzymes of term placental cytosol were studied using a dextran-coated charcoal (DCC) adsorption assay as a rapid screen for GnRH tracer inactivation. Three different activities were demonstrable: (i) a cathepsin D-like enzyme (M(r) 55 kDa), active against all radiolabelled GnRH isoforms and agonists tested, optimal at acid pH, and inhibited specifically by pepstatin; (ii) a metallo-thiol endopeptidase activity (M(r) 70 kDa) optimal at alkaline pH (7-9) which degraded GnRH isoforms to a greater extent than GnRH analogues, inhibited dose-dependently by low concentrations of thiol reagents (N-ethylmaleimide, thimerosal), chelating agents (o-phenanthroline, EDTA), and by tosyl-phenylalanyl-chloromethyl ketone but not by other serine protease inhibitors; and (iii) a bacitracin-sensitive enzyme optimal at physiological pH. These observations permitted the development of a robust radioreceptor assay which minimized GnRH tracer degradation. Under these assay conditions, the GnRH-like radioreceptor assay activity of human placental cytosol fractions was markedly reduced.
Bramley
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The University of Edinburgh Department of Obstetrics and Gynaecology, Centre for Reproductive Biology, 37 Chalmers Street, Edinburgh EH3 9EW, UK.
Menzies
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D000327:Adsorption; D000818:Animals; D002402:Cathepsin D / Q000378:metabolism; D002606:Charcoal; D002645:Chickens; D002850:Chromatography, Gel; D003600:Cytosol / Q000378:metabolism; D003911:Dextrans; D005260:Female; D007987:Gonadotropin-Releasing Hormone / Q000037:antagonists & inhibitors / Q000302:isolation & purification / Q000378:metabolism*; D006801:Humans; D006863:Hydrogen-Ion Concentration; D066298:In Vitro Techniques; D010447:Peptide Hydrolases / Q000378:metabolism*; D010920:Placenta / Q000378:metabolism*; D011247:Pregnancy; D011480:Protease Inhibitors / Q000494:pharmacology; D020033:Protein Isoforms / Q000378:metabolism; D011869:Radioligand Assay; D051381:Rats; D017207:Rats, Sprague-Dawley
D003911:Dextrans; D011480:Protease Inhibitors; D020033:Protein Isoforms; D002606:Charcoal; D007987:Gonadotropin-Releasing Hormone; D010447:Peptide Hydrolases; D002402:Cathepsin D