Covalent crosslinking of angiotensin II to its binding sites in rat adrenal membranes.

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Abstract
To identify the molecular components of the angiotensin II (AII) receptor in the adrenal cortex, 125I-labeled AII (125I-AII) was covalently linked to its binding sites by using the chemical crosslinker disuccinimidyl suberate. Membrane fractions from rat adrenal tissue were incubated with 125I-AII, washed, and treated with disuccinimidyl suberate. NaDodSO4 polyacrylamide gel electrophoresis carried out under reducing conditions followed by autoradiography showed that a major polypeptide(s) with an average MW of 116,000 was covalently tagged with 125I-AII. Proteins with MW 54,000 and 45,000 were also lightly labeled. Labeling of the MW 116,000 species was specific in that it could be abolished by prior incubation of the membranes with [Sar1,Leu8]AII or unlabeled AII. Labeling of the smaller proteins was less affected by prior incubation with [Sar1,Leu8]AII. Experiments in which crosslinking was carried out in the presence of a variety of protease inhibitors indicated that the MW 116,000 species is not an enzyme involved in AII degradation. Gel electrophoresis under nonreducing conditions showed that this component is not derived from large disulfide-linked entities. The MW 116,000 moiety apparently is a part of the receptor system for AII. The relationship, if any, of the MW 54,000 and 45,000 peptides to the AII receptor remains unknown.