Purification of the hepatic vasopressin receptor using a novel affinity column.
A vasopressin receptor was purified, using a novel affinity column, from rat liver plasma membranes treated with guanosine 5'-(3-O-thio)triphosphate and solubilized with 0.8% cholate. Incubation of the membranes with the GTP analogue resulted in a dissociation of the receptor-guanine nucleotide regulatory protein complex. This manipulation, although resulting in a low-affinity state of the receptor, facilitated purification. The solubilized receptor was assayed using a new reconstitution procedure in which the soluble extracts were inserted into lipid vesicles composed of phosphatidylcholine and phosphatidylinositol. The receptor was purified by sequential chromatography on Q-Sepharose and hydroxyapatite. The use of a novel affinity column, a V1-vasopressin antagonist-agarose, resulted in a near-homogeneous preparation of a protein which exhibited an Mr = 58,000 on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Autoradiography of purified receptor, as well as crude membrane preparations cross-linked to [125I]arginine vasopressin, also revealed a protein band with an approximate Mr = 58,000. These findings indicate that V1-antagonist affinity chromatography should be useful for purifying adequate amounts of the receptor for studies of structure and function.
Georgoussi
Z
Z
Howard Hughes Medical Institute, Vanderbilt University School of Medicine, Nashville, TN 37232.
Taylor
S J
SJ
Bocckino
S B
SB
Exton
J H
JH
D000818:Animals; D002462:Cell Membrane / Q000737:chemistry; D019826:Cholic Acid; D002793:Cholic Acids; D002846:Chromatography, Affinity; D003432:Cross-Linking Reagents; D003902:Detergents; D017886:Durapatite; D006493:Heparin; D006882:Hydroxyapatites; D008099:Liver / Q000737:chemistry*; D008567:Membranes, Artificial; D051381:Rats; D011945:Receptors, Angiotensin / Q000302:isolation & purification*; D017483:Receptors, Vasopressin; D012685:Sepharose / Q000031:analogs & derivatives; D012995:Solubility; D014667:Vasopressins / Q000378:metabolism*
D002793:Cholic Acids; D003432:Cross-Linking Reagents; D003902:Detergents; D006882:Hydroxyapatites; D008567:Membranes, Artificial; D011945:Receptors, Angiotensin; D017483:Receptors, Vasopressin; D014667:Vasopressins; D006493:Heparin; D012685:Sepharose; D017886:Durapatite; D019826:Cholic Acid