Reconstitution of the myometrial oxytocin receptor into proteoliposomes. Dependence of oxytocin binding on cholesterol.
The requirements for regaining high-affinity binding of the myometrial oxytocin receptor after detergent solubilization were investigated by reconstitution experiments. Large unilamellar liposomes were prepared by reverse-phase evaporation from different mixtures of phospholipids, cholesterol and cholesteryl hemisuccinate. In the presence of the oxytocin receptor solubilized from myometrial membranes from pregnant guinea pig uterus, liposomes were treated with 3-[(3-cholamidopropyl)-dimethylammonio]-2-hydroxy-1-propanesulfonate (Chapso) throughout the range of detergent concentrations that cause the transformation of lamellar structures to mixed micelles. Detergent removal was achieved using bio-beads SM-2 as adsorbent. The presence of cholesterol was a prerequisite for regaining high-affinity binding of [3H]oxytocin and 125I-oxytocin antagonist to reconstituted proteoliposomes. Binding of [3H]oxytocin but not of the antagonist was dependent on the presence of Mn2+ ions. Reconstitution after lectin chromatography and photoaffinity labeling of reconstituted vesicles resulted in the exclusive labeling of the oxytocin receptor with a molecular mass of 68-80 kDa.
Klein
U
U
Max-Planck-Institut für Biophysik, Frankfurt, Germany.
Fahrenholz
F
F
D000345:Affinity Labels; D000818:Animals; D002784:Cholesterol / Q000494:pharmacology*; D002793:Cholic Acids; D003902:Detergents; D004591:Electrophoresis, Polyacrylamide Gel; D005260:Female; D006168:Guinea Pigs; D007700:Kinetics; D008081:Liposomes; D008823:Micelles; D008970:Molecular Weight; D009215:Myometrium / Q000378:metabolism*; D010121:Oxytocin / Q000378:metabolism*; D011247:Pregnancy; D011510:Proteolipids / Q000378:metabolism*; D018045:Receptors, Oxytocin / Q000187:drug effects / Q000302:isolation & purification / Q000378:metabolism*
D000345:Affinity Labels; D002793:Cholic Acids; D003902:Detergents; D008081:Liposomes; D008823:Micelles; D011510:Proteolipids; D018045:Receptors, Oxytocin; C020485:proteoliposomes; D010121:Oxytocin; C048531:chapso; D002784:Cholesterol