Solubilization and molecular characterization of active galanin receptors from rat brain.
Galanin receptors were solubilized from rat brain using the zwitterionic detergent 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid (CHAPS). Binding of 125I-galanin to the soluble fraction was time- and temperature-dependent, saturable, and reversible. Scatchard analysis of binding data indicated that the soluble extract contained a single class of galanin binding sites with a Kd of 0.8 nM and a Bmax of 26 fmol/mg of protein. Unlabeled galanin and its fragments galanin(2-29) and galanin(1-15) antagonized the binding of 125I-galanin to CHAPS-solubilized extracts with relative potencies similar to those observed with membrane receptors. Galanin(3-29) was found inactive. Binding of 125I-galanin to CHAPS extracts was inhibited by guanine nucleotides with the following rank order of potency: GMP-P-(NH)P greater than GTP greater than GDP. Molecular analysis of the soluble galanin receptor by covalent cross-linking of 125I-galanin to CHAPS extracts using disuccinimidyl tartrate and further identification on SDS-PAGE indicated that the soluble galanin binding site behaves as a protein of Mr 54,000. After incubation of CHAPS extracts with 125I-galanin, gel filtration on Sephacryl S-300 followed by ultracentrifugation on sucrose density gradient revealed a binding component with the following hydrodynamic parameters: Stokes radius, 5 nm; s20,w, 4.5 S; Mr, 98,000; frictional ratio, 1.6. GMP-P(NH)P treatment of CHAPS extracts gave rise to a molecular form with the following characteristics: Stokes radius, 4 nm; s20,w, 3.3 S; Mr, 57,000; frictional ratio, 1.4.(ABSTRACT TRUNCATED AT 250 WORDS)
Chen
Y
Y
Laboratoire de Biologie et Physiologie des Cellules Digestives, Institut National de la Santé et de la Recherche Médicale, U 239, Paris, France.
Couvineau
A
A
Laburthe
M
M
Amiranoff
B
B
D000818:Animals; D001667:Binding, Competitive; D001923:Brain Chemistry; D002462:Cell Membrane / Q000737:chemistry; D002793:Cholic Acids; D003902:Detergents; D019204:GTP-Binding Proteins / Q000737:chemistry; D019004:Galanin; D007700:Kinetics; D008297:Male; D008970:Molecular Weight; D009479:Neuropeptides / Q000737:chemistry*; D010455:Peptides / Q000737:chemistry*; D011485:Protein Binding; D051381:Rats; D011919:Rats, Inbred Strains; D044088:Receptors, Galanin; D011964:Receptors, Gastrointestinal Hormone / Q000737:chemistry* / Q000187:drug effects; D012995:Solubility; D013329:Structure-Activity Relationship
D002793:Cholic Acids; D003902:Detergents; D009479:Neuropeptides; D010455:Peptides; D044088:Receptors, Galanin; D011964:Receptors, Gastrointestinal Hormone; D019004:Galanin; D019204:GTP-Binding Proteins; C028213:3-((3-cholamidopropyl)dimethylammonium)-1-propanesulfonate