Solubilization and characterization of active neuropeptide Y receptors
from rabbit kidney
J Biol Chem, 265 (30):
18142-7 (October 1990)Gimpl, G Gerstberger, R Mauss, U Klotz, K N Lang, R E Research Support,
Non-U.S. Gov't United states The Journal of biological chemistry
J Biol Chem. 1990 Oct 25;265(30):18142-7..Abstract
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.