Solubilization and partial characterization of angiotensin II receptors from rat brain.
Rat brain angiotensin II (Ang II) receptors were solubilized with a yield of 30-40% using the synthetic detergent 3[(3-cholamidopropyl)dimethylammonio)]-1-propanesulfonate. Kinetic analysis employing the high-affinity antagonist 125I-Sar1,Ile8-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 pIC50 values of a series of agonist and antagonists competing for 125I-Sar1,Ile8-Ang II labeled binding sites in either solubilized or intact membranes. Moreover, covalent labeling of 125I-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 125I-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 125I-Sar1,Ile8-Ang II prior to gel filtration, and chromatofocusing of antagonist labeled sites revealed only one peak with an isoelectric point of 6.2. The successful solubilization of these binding sites should facilitate continued investigation of Ang II receptors in the brain.
Siemens
I R
IR
Department of Animal Biology, University of Pennsylvania, Philadelphia 19104-6046.
Swanson
G N
GN
Fluharty
S J
SJ
Harding
J W
JW
D000804:Angiotensin II / Q000031:analogs & derivatives / Q000378:metabolism* / Q000494:pharmacology; D000818:Animals; D001667:Binding, Competitive; D001921:Brain / Q000378:metabolism*; D002462:Cell Membrane / Q000378:metabolism; D003432:Cross-Linking Reagents / Q000494:pharmacology; D007700:Kinetics; D008297:Male; D011869:Radioligand Assay; D051381:Rats; D011919:Rats, Inbred Strains; D011945:Receptors, Angiotensin / Q000187:drug effects / Q000302:isolation & purification / Q000378:metabolism*; D012995:Solubility; D013388:Succinimides / Q000494:pharmacology
D003432:Cross-Linking Reagents; D011945:Receptors, Angiotensin; D013388:Succinimides; D000804:Angiotensin II; C019358:disuccinimidyl suberate