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Solubilization of the Neuropeptide Y Receptor from Rat Brain Membranes
Author(s) -
Man Peter J.,
Mervin Sabrena J.,
Taylor Ian L.
Publication year - 1991
Publication title -
journal of neurochemistry
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.75
H-Index - 229
eISSN - 1471-4159
pISSN - 0022-3042
DOI - 10.1111/j.1471-4159.1991.tb02084.x
Subject(s) - chaps , neuropeptide y receptor , peptide yy , receptor , chemistry , membrane , gel electrophoresis , biochemistry , g protein , pancreatic polypeptide , peptide , sodium dodecyl sulfate , chromatography , neuropeptide , glucagon , hormone
The neuropeptide Y (NPY) receptor was solubilized from rat brain membranes with the zwitterionic detergent 3‐[(3‐cholamidopropyl)‐dimethylammonio]‐1‐propanesulfonate (CHAPS). The binding of 125 I‐NPY to CHAPS extracts was protein, time, and temperature dependent. Unlabeled NPY and the related peptides peptide YY (PYY) and pancreatic polypeptide inhibited 125 I‐NPY binding to solubilized receptors with relative potencies similar to those seen with membrane‐bound receptors: NPY > PYY ± pancreatic polypeptide. Scatchard analysis of equilibrium binding data showed the CHAPS extracts to contain a single population of binding sites with a K D of 3.6 ± 0.4 n M (mean ± SEM) and a B max of 5.0 ± 0.2 pmol/mg of protein. In addition the 125 I‐NPY binding to the soluble receptor was not inhibited by guanosine‐5′‐ O ‐(3‐thiotriphosphate), in contrast to the GTP sensitivity displayed by the membrane‐bound receptor. Gel filtration chromatography using Sepharose 6B revealed a single peak of binding activity corresponding to a M r of ∼67,000, and sodium dodecyl sulfate‐polyacrylamide gel electrophoresis analysis after chemical cross‐linking revealed a single band at M r 62,000. After solubilization and gel chromatography a 50‐ to 100‐fold purification of the NPY receptor was obtained.