Solubilization and characterization of an angiotensin II binding protein from liver.
Binding sites with high affinity for angiotensin II were solubilized from hepatic membranes by treatment with digitonin. Binding of radioiodinated angiotensin II was assayed by gel filtration and independently by a technique exploiting the failure of activated charcoal to adsorb the bound ligand. The binding protein was partially purified using ammonium sulfate fractionation followed by gel filtration, and in the presence of protease inhibitors, the isolated binding protein preparation did not catalyze degradation of the angiotensin II. Binding to the membranes as well as to the solubilized preparation was specific and saturable. The membranes exhibited a single set of high-affinity binding sites with a Kd of 0.5 nM. The solubilized preparation, also showed the presence of a single class of high-affinity binding sites (Kd = 10.5 nM). Displacement studies using angiotensin I as well as various fragments, agonists and antagonists of angiotensin II disclosed a structure-activity profile similar to that found with intact membranes. Dissociation of angiotensin II from the soluble macromolecular complex was slow but was enhanced at non-physiological pH values or in the presence of 4.5 M urea, or 1% sodium dodecyl sulfate. Covalent binding of the radioiodinated angiotensin II to a single, specific macromolecular component was achieved by treatment with disuccinimidyl suberate. The apparent molecular weight of this reduced, denatured radioactive protein was estimated at about 68 000 by polyacrylamide gel electrophoresis.
D000804:Angiotensin II; D000818:Animals; D002850:Chromatography, Gel; D003902:Detergents; D007700:Kinetics; D008099:Liver / Q000378:metabolism*; D008297:Male; D008970:Molecular Weight; D011817:Rabbits; D011945:Receptors, Angiotensin / Q000302:isolation & purification*; D011956:Receptors, Cell Surface / Q000302:isolation & purification*; D012995:Solubility
D003902:Detergents; D011945:Receptors, Angiotensin; D011956:Receptors, Cell Surface; D000804:Angiotensin II