Solubilization of the neuropeptide Y receptor from rat brain membranes.
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 125I-NPY to CHAPS extracts was protein, time, and temperature dependent. Unlabeled NPY and the related peptides peptide YY (PYY) and pancreatic polypeptide inhibited 125I-NPY binding to solubilized receptors with relative potencies similar to those seen with membrane-bound receptors: NPY greater than PYY much greater than pancreatic polypeptide. Scatchard analysis of equilibrium binding data showed the CHAPS extracts to contain a single population of binding sites with a KD of 3.6 +/- 0.4 nM (mean +/- SEM) and a Bmax of 5.0 +/- 0.2 pmol/mg of protein. In addition the 125I-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 Mr of approximately 67,000, and sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis after chemical cross-linking revealed a single band at Mr 62,000. After solubilization and gel chromatography a 50- to 100-fold purification of the NPY receptor was obtained.
Mannon
P J
PJ
Division of Gastroenterology, Duke University Medical Center, Durham, North Carolina.
Mervin
S J
SJ
Taylor
I L
IL
D000818:Animals; D001921:Brain / Q000378:metabolism*; D002793:Cholic Acids; D002850:Chromatography, Gel; D003432:Cross-Linking Reagents; D003902:Detergents; D008297:Male; D008566:Membranes / Q000378:metabolism; D009478:Neuropeptide Y / Q000378:metabolism; D051381:Rats; D011919:Rats, Inbred Strains; D017476:Receptors, Neuropeptide Y; D017981:Receptors, Neurotransmitter / Q000378:metabolism*; D012995:Solubility; D014020:Tissue Extracts / Q000378:metabolism
D002793:Cholic Acids; D003432:Cross-Linking Reagents; D003902:Detergents; D009478:Neuropeptide Y; D017476:Receptors, Neuropeptide Y; D017981:Receptors, Neurotransmitter; D014020:Tissue Extracts; C028213:3-((3-cholamidopropyl)dimethylammonium)-1-propanesulfonate