Evidence of essential disulfide bonds in angiotensin II binding sites of rabbit hepatic membranes. Inactivation by dithiothreitol.
Radiolabelled angiotensin II binds to a single class of high-affinity binding sites on purified rabbit hepatic membranes. The binding is specific, reversible and saturable. Displacement studies using angiotensin and various analogs of angiotensin II disclosed a structure-activity profile similar to that found in physiologically relevant angiotensin II receptor sites. Treatment of membranes with the reducing agent, dithiothreitol, causes a significant decrease in the affinity of angiotensin II binding sites for the native ligand. This effect is mimicked by a 15-fold higher concentration of the monosulfhydryl derivative, 2-mercaptoethanol. Kinetic studies also indicated that dithiothreitol increases the rate of dissociation of bound ligand from the membrane without significantly affecting the association rate. In contrast, treatment of membranes with the metal chelators, ethylenediaminetetracetic acid (EDTA) and ethyleneglycol bis(beta-aminoethyl ether)-N,N'-tetracetic acid (EGTA), does not affect the binding of radiolabeled angiotensin II. Furthermore, dithiothreitol inhibited the binding of angiotensin II to a solubilized partially purified preparation of angiotensin II-binding protein from the same tissue and also increased the dissociation of bound angiotensin II. This indicates that the effect of the sulfhydryl reagents on the membrane binding sites is the result of a direct alteration of the binding sites rather than a gross modification of the structure of the membrane.
D000804:Angiotensin II / Q000378:metabolism*; D000818:Animals; D001665:Binding Sites; D002462:Cell Membrane / Q000187:drug effects / Q000378:metabolism*; D004072:Digitonin; D004220:Disulfides / Q000032:analysis*; D004229:Dithiothreitol / Q000494:pharmacology*; D006863:Hydrogen-Ion Concentration; D008099:Liver / Q000648:ultrastructure*; D008274:Magnesium / Q000494:pharmacology; D008623:Mercaptoethanol / Q000494:pharmacology; D011817:Rabbits; D011945:Receptors, Angiotensin / Q000378:metabolism; D012995:Solubility; D013696:Temperature
D004220:Disulfides; D011945:Receptors, Angiotensin; D000804:Angiotensin II; D008623:Mercaptoethanol; D008274:Magnesium; D004072:Digitonin; D004229:Dithiothreitol