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Molecular Human Reproduction, Vol. 6, No. 2, 113-126, February 2000
© 2000 European Society of Human Reproduction and Embryology

Endocrinology

Human placental gonadotrophin-releasing hormone-like factors: an artefact of human placental peptidases?

T.A. Bramley1 and G.S. Menzies

The University of Edinburgh Department of Obstetrics and Gynaecology, Centre for Reproductive Biology, 37 Chalmers Street, Edinburgh EH3 9EW, UK

Abstract

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 125I-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 (Mr 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 (Mr 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.

GnRH receptor/peptidases/placenta

Notes

1 To whom correspondence should be addressed


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This article has been cited by other articles:


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T.A. Bramley, K. Campbell, and G.S. Menzies
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