Solubilization and characterization of active neuropeptide Y receptors from rabbit kidney.
Active neuropeptide Y receptors were solubilized from rabbit kidney membranes using the zwitterionic detergent 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid (CHAPS). In membrane fragments and soluble extracts neuropeptide Y binding was time dependent, saturable, reversible, and of high affinity. Scatchard analysis of equilibrium binding data indicated a single class of binding sites with respective KD and Bmax values of 0.09 nM and 530 fmol/mg of protein for the membrane-bound receptors and 0.10 nM and 1585 fmol/mg of protein for the soluble receptors. Neuropeptide Y binding was specifically inhibited by the nonhydrolyzable GTP analog guanosine 5'-O-(3-thiotriphosphate) in a concentration-dependent manner, with IC50 values of 28 and 0.14 microM for membrane-bound and soluble receptors, respectively, suggesting that neuropeptide Y receptors are functionally coupled to GTP-binding regulatory proteins. Cross-linking studies were performed with the heterobifunctional N-hydroxysuccinimidyl-4-azidobenzoate and the monofunctional neuropeptide Y derivative, azidobenzoyl and led to the identification of a 100 kDa peptide that should represent the covalently labeled neuropeptide Y receptor.
Gimpl
G
G
Department of Pharmacology, University of Heidelberg, Federal Republic of Germany.
Gerstberger
R
R
Mauss
U
U
Klotz
K N
KN
Lang
R E
RE
D000345:Affinity Labels; D000818:Animals; D002462:Cell Membrane / Q000737:chemistry; D002793:Cholic Acids; D002851:Chromatography, High Pressure Liquid; D003902:Detergents; D007668:Kidney / Q000737:chemistry*; D009478:Neuropeptide Y; D011817:Rabbits; D017476:Receptors, Neuropeptide Y; D017981:Receptors, Neurotransmitter / Q000302:isolation & purification*; D012995:Solubility
D000345:Affinity Labels; D002793:Cholic Acids; D003902:Detergents; D009478:Neuropeptide Y; D017476:Receptors, Neuropeptide Y; D017981:Receptors, Neurotransmitter; C028213:3-((3-cholamidopropyl)dimethylammonium)-1-propanesulfonate