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Solubilization and Partial Characterization of Angiotensin II Receptors from Rat Brain
Author(s) -
Siemens I. R.,
Swanson G. N.,
Fluharty S. J.,
Harding J. W.
Publication year - 1991
Publication title -
journal of neurochemistry
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.75
H-Index - 229
eISSN - 1471-4159
pISSN - 0022-3042
DOI - 10.1111/j.1471-4159.1991.tb03801.x
Subject(s) - chemistry , receptor , membrane , gel electrophoresis , angiotensin ii , size exclusion chromatography , agonist , biochemistry , sodium dodecyl sulfate , enzyme
Rat brain angiotensin II (Ang II) receptors were solubilized with a yield of 30–40% using the synthetic detergent 3[(3‐cholamidopropyl)dimethylammonio)]‐1 ‐propane‐sulfonate. Kinetic analysis employing the high‐affinity antagonist I25 I‐Sar 1 , lle 8 ‐Ang II indicated that the solubilized receptors exhibited the same properties as receptors present within intact brain membranes. Furthermore, there was a positive correlation ( r − 0.99) between the respective pIC 50 values of a series of agonist and antagonists competing for 125 I‐Sar l ,IIe 8 ‐Ang II labeled binding sites in either solubilized or intact membranes. Moreover, covalent labeling of 125 I‐ Ang II to solubilized receptors with the homo‐bifunctional cross‐linker disuccinimidyl suberate, followed by gel filtration, revealed one major and one minor binding peak with apparent molecular weights of 64,000 and 115,000, respectively. Two binding proteins of comparable molecular weights (i.e., 112,000 and 60,000) were also identified by covalent cross‐linking of I25 l‐Ang II to solubilized brain membranes followed by sodium dodecyl sulfate‐polyacrylamide gel electrophoresis analysis. In contrast, only the smaller molecular mass binding protein was observed when solubilized membranes were labeled with the antagonist 125 I‐Sar 1 .IIe 8 ‐Ang II prior to gel filtration, and chromatofocusing of antagonist labeled sites revealed only one peak with an isodectric point of 6.2. The successful solubilization of these binding sites should facilitate continued investigation of Ang II receptors in the brain.