Crosslinking analysis of an endothelin receptor protein from human placenta.
Crosslinking of [125I]ET-1 to human placental membrane preparations in the absence of divalent cations revealed a [125I]ET-1.ET-1-receptor complex of 49 kDa and of 205 kDa on SDS-PAGE. In the presence of 20 mM MnCl2, however, only a complex of 205 kDa was observed. About 64% of the radioactively labeled receptor.ligand complex could be solubilised by digitonin and CHAPS. Size exclusion chromatography of the solubilised placental membranes, prelabeled with [125I]ET-1 in the presence of MnCl2, revealed two binding proteins of 200 and 33 kDa. In the absence of MnCl2, however, only a binding protein of 50 kDa was observed. Binding of [125I]ET-1 to the 200 kDa and 50 kDa but not to the 33 kDa protein was suppressed by unlabeled ET-1. Moreover the ET-1-receptor density in placental membranes (Bmax) increased significantly in the presence of MnCl2, whereas the dissociation constant (Kd) decreased. Thus, divalent cations exhibit marked and possibly physiologically important effects on molecular mass, Bmax and Kd of the [125I]ET-1.ET-1-receptor complex. The potential mechanism of action of Mn2+ ions on the ET-1-receptor is discussed.
Kalina
B
B
Pharma Division, F. Hoffmann-La Roche Ltd, Basel, Switzerland.
Löffler
B M
BM
D002413:Cations, Divalent / Q000737:chemistry; D002712:Chlorides; D003432:Cross-Linking Reagents; D004591:Electrophoresis, Polyacrylamide Gel; D016232:Endothelins / Q000737:chemistry / Q000378:metabolism*; D005260:Female; D006801:Humans; D008345:Manganese / Q000494:pharmacology; D017895:Manganese Compounds; D008970:Molecular Weight; D010920:Placenta / Q000378:metabolism*; D011247:Pregnancy; D017466:Receptors, Endothelin / Q000737:chemistry / Q000378:metabolism*; D012995:Solubility
D002413:Cations, Divalent; D002712:Chlorides; D003432:Cross-Linking Reagents; D016232:Endothelins; D017895:Manganese Compounds; D017466:Receptors, Endothelin; D008345:Manganese; C025340:manganese chloride