Human placental gonadotrophin-releasing hormone (GnRH) binding sites: II. Comparison of binding and inactivation of 125I-labelled GnRH agonist to subcellular fractions following density gradient centrifugation.
Human placental homogenates and membrane fractions were centrifuged on continuous sucrose density gradients, with or without buoyant density perturbation of plasma-membranes by digitonin, and aliquots of each gradient fraction were assayed for a range of plasma-membrane and intracellular organelle markers, and for specific binding and inactivation of radiolabelled GnRH agonist (GnRHA), Buserelin ([D-Ser(tBu)6] GnRH 1-9 ethylamide). GnRH agonist (GnRHA) binding equilibrated in the same regions of control gradients as the plasma-membrane markers, EGF-receptor and alkaline phosphatase. Moreover, binding of 125I-labelled GnRHA and 125I-labelled chicken GnRH II (ch GnRH II) was enriched in the same regions of the gradients, indicating that both bound to the same membrane fractions. Digitonin pretreatment increased the buoyant density of all three placental plasma-membrane markers to a similar degree. Intracellular organelle markers (and hCG content) equilibrated in different regions of the gradient to placental surface-membranes, and were not perturbed appreciably by digitonin. In contrast, inactivation of 125I-labelled GnRHA was associated largely with the soluble (cytosol) fraction which failed to enter the gradient, and little tracer inactivation was observed in fractions enriched in GnRHA binding activity. Similar results were obtained with fractionated rat pituitary membranes. We conclude that: (a) placental GnRH binding sites do not represent binding of radiolabelled ligand to GnRH-degrading enzymes, (b) degradation of radiolabelled ligand is associated largely with placental cytosol fractions, and (c) GnRH binding activity appears to be associated largely with placental plasma-membranes.
Menzies
G S
GS
University of Edinburgh Department of Obstetrics & Gynaecology, Scotland.
Bramley
T A
TA
D000818:Animals; D002499:Centrifugation, Density Gradient; D005591:Chemical Fractionation; D005260:Female; D007987:Gonadotropin-Releasing Hormone / Q000031:analogs & derivatives* / Q000037:antagonists & inhibitors / Q000378:metabolism; D006801:Humans; D007457:Iodine Radioisotopes; D010902:Pituitary Gland / Q000378:metabolism; D010920:Placenta / Q000378:metabolism*; D011247:Pregnancy; D011257:Pregnancy Proteins / Q000378:metabolism*; D011869:Radioligand Assay; D051381:Rats; D017207:Rats, Sprague-Dawley; D011966:Receptors, LHRH / Q000378:metabolism*; D013347:Subcellular Fractions / Q000378:metabolism; D017329:Triptorelin Pamoate / Q000031:analogs & derivatives*
D007457:Iodine Radioisotopes; D011257:Pregnancy Proteins; D011966:Receptors, LHRH; D017329:Triptorelin Pamoate; D007987:Gonadotropin-Releasing Hormone; C019176:Tryptal