Purification of a galanin receptor from pig brain.
A galanin receptor protein was solubilized with 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate (CHAPS) from pig brain membranes and then purified by single-step affinity chromatography. The product exhibits saturable and specific binding for galanin with a binding activity of 17 nmol/mg of protein and a dissociation constant (Kd) of 10 nM. This represents a 300,000-fold purification over the detergent-solubilized fraction with a final recovery of 31% of the initial membrane galanin binding activity. Gel electrophoresis of the affinity-purified material showed a single polypeptide of 54 kDa by silver staining and after radioiodination. Cross-linking of a purified fraction affinity-labeled with 125I-labeled galanin revealed a single band for the galanin-receptor complex at 57 kDa. The general binding characteristics of the purified preparation appeared to be identical to those of the crude soluble material as far as specificity toward galanin and the structural requirement for galanin are concerned. In contrast, unlike the CHAPS-soluble galanin receptor, binding of 125I-labeled galanin to the purified galanin receptor was not sensitive to guanine nucleotides, suggesting that dissociation of the inhibitory guanine nucleotide binding protein from the galanin receptor occurred during purification. The purification to homogeneity of a galanin receptor paves the way toward its sequencing and cloning.
Chen
Y
Y
Laboratoire de Biologie and Physiologie des Cellules Digestives, Institut National de la Santé et de la Recherche Médicale, U 239, Paris, France.
Fournier
A
A
Couvineau
A
A
Laburthe
M
M
Amiranoff
B
B
D000818:Animals; D001345:Autoradiography; D001667:Binding, Competitive; D001921:Brain / Q000378:metabolism*; D002462:Cell Membrane / Q000378:metabolism; D002793:Cholic Acids; D002846:Chromatography, Affinity; D004591:Electrophoresis, Polyacrylamide Gel; D019004:Galanin; D007457:Iodine Radioisotopes; D007700:Kinetics; D008970:Molecular Weight; D009479:Neuropeptides / Q000378:metabolism*; D010455:Peptides / Q000378:metabolism*; D044088:Receptors, Galanin; D011964:Receptors, Gastrointestinal Hormone / Q000302:isolation & purification* / Q000378:metabolism*; D013552:Swine
D002793:Cholic Acids; D007457:Iodine Radioisotopes; D009479:Neuropeptides; D010455:Peptides; D044088:Receptors, Galanin; D011964:Receptors, Gastrointestinal Hormone; D019004:Galanin; C028213:3-((3-cholamidopropyl)dimethylammonium)-1-propanesulfonate