Isolation and characterization of imidazoline receptor protein from bovine adrenal chromaffin cells.
We sought to isolate and partially purify proteins corresponding to the binding element of the imidazoline receptor (IR) from adrenal chromaffin cell membranes. These cells express IRs of the I-2 subclass and not alpha 2-adrenergic receptors. Proteins were solubilized in 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate-containing buffer and were assayed by binding of [3H]idazoxan, an imidazoline radioligand. Two ligand affinity resins, p-aminoclonidine-Trisacryl GF-2000 (PAC-ReactiGel) and idazoxan-PharmaLink agarose (IDA-agarose), were synthesized. These allowed purification by single-step affinity chromatography of a major receptor binding protein component of 70 kDa, as assessed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis and [3H]idazoxan binding assay. The purified imidazoline-binding proteins from IDA-agarose and PAC-ReactiGel had similar affinities for the radioligand [3H]idazoxan (Kd = 3.7 and 4.9 nM, respectively) and a displacement profile, showing sensitivity to imidazoline agents (cirazoline > clonidine) and insensitivity to catecholamines and adrenergic agents (epinephrine approximately rauwolscine), that was similar to that of the intact membrane receptor. The imidazoline-binding protein did not bind to concanavalin A, suggesting that it may not be glycosylated or that the sugar moieties present are not recognized by this lectin. The results indicate that IR and alpha 2 receptor proteins may be biochemically distinct and that IDA-agarose and PAC-ReactiGel columns are useful for purification of sufficient quantities of imidazoline-binding proteins to allow for structural and functional studies of the IR.
Wang
H
H
Department of Neurology and Neuroscience, Cornell University Medical College, New York, New York 10021.
Regunathan
S
S
Meeley
M P
MP
Reis
D J
DJ
D000311:Adrenal Glands / Q000378:metabolism*; D000818:Animals; D001710:Biotin / Q000378:metabolism; D002417:Cattle; D002837:Chromaffin Granules / Q000378:metabolism*; D002846:Chromatography, Affinity; D003208:Concanavalin A / Q000378:metabolism; D004146:Dioxanes / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel; D019329:Idazoxan; D054755:Imidazoline Receptors; D008565:Membrane Proteins / Q000378:metabolism*; D011955:Receptors, Drug / Q000302:isolation & purification* / Q000378:metabolism; D016622:Silver Staining
D004146:Dioxanes; D054755:Imidazoline Receptors; D008565:Membrane Proteins; D011955:Receptors, Drug; D003208:Concanavalin A; D001710:Biotin; D019329:Idazoxan