Physicochemical characterization of photoaffinity-labeled angiotensin II receptors.
Specific receptor sites for angiotensin II (AII) were analyzed in the adrenal cortex and other target tissues including liver, anterior pituitary gland, and smooth muscle, after covalent labeling with the radioactive photoaffinity analog 125I-[Sar1,(4-N3)Phe8]-AII. The photoreactive AII derivative retained high affinity for adrenal receptors and full steroidogenic activity in adrenal glomerulosa cells. In bovine adrenal cortex, covalent labeling of AII receptors by the photoreactive analog was specifically inhibited by [Sar1]AII with an IC50 of about 5 nM. The Mr of the covalent AII-receptor complex during polyacrylamide gel electrophoresis of the labeled protein under reducing conditions was 58,000. Under non-reducing conditions, a minor band with Mr of 105,000 was also observed. Two labeled species were also found during gel permeation chromatography of the detergent-solubilized complex, with Mrs of 64,000 and 86,000. The pl of the solubilized photolabeled complex was absorbed to wheat germ lectin Sepharose 6MB and could be eluted by N-acetylglucosamine. The Mrs of specific AII-binding sites in several target tissues, determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, showed target tissues, determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, showed significant differences within and between species. The most striking differences were between rat adrenal cortex (79,000) and both rat liver (60,000) and bovine adrenal cortex (58,000). After enzymatic deglycosylation, the Mr of the major component present in the bovine and rat adrenal cortex decreased by 40% and 55% to 35,000 and 34,000, respectively, suggesting that variations in carbohydrate content contribute to the physical heterogeneity of AII receptors in individual target tissues.
D000302:Adrenal Cortex / Q000648:ultrastructure; D000345:Affinity Labels / Q000378:metabolism*; D000804:Angiotensin II / Q000031:analogs & derivatives*; D000818:Animals; D001667:Binding, Competitive; D002417:Cattle; D055598:Chemical Phenomena; D002621:Chemistry; D002850:Chromatography, Gel; D004591:Electrophoresis, Polyacrylamide Gel; D006026:Glycoside Hydrolases / Q000494:pharmacology; D000882:Haplorhini; D007525:Isoelectric Focusing; D008027:Light; D008099:Liver / Q000648:ultrastructure; D009130:Muscle, Smooth / Q000648:ultrastructure; D009420:Nervous System / Q000648:ultrastructure; D011817:Rabbits; D051381:Rats; D011945:Receptors, Angiotensin / Q000302:isolation & purification* / Q000378:metabolism
D000345:Affinity Labels; D011945:Receptors, Angiotensin; D000804:Angiotensin II; C056961:angiotensin II, Sar(1)-4-azido-Phe(8)-; D006026:Glycoside Hydrolases