Purification and biochemical characterization of neuropeptide Y2 receptor.
Neuropeptide Y (NPY) receptors consist of three subtypes, designated NPY1, NPY2, and NPY3. The Y1 receptor has been cloned. The present study reports the purification of the NPY-Y2 receptor from porcine brain and its biochemical characterization. NPY receptors were solubilized and purified by sequential hydrophobic interaction, ion exchange, and NPY-affinity chromatography. By use of SDS-polyacrylamide gel electrophoresis, high performance liquid chromatography gel permeation chromatography, and chemical cross-linking studies, the affinity-purified brain NPY-Y2 receptor was identified as a monomeric glycoprotein with a molecular mass of 60 kDa. Following deglycosylation, the molecular mass of the Y2 receptor was decreased to 45 kDa. Although the 125I-NPY binding to the purified NPY receptor was considerably decreased by N-ethylmaleimide, guanine nucleotides had no effect. Therefore, the purified NPY-Y2 receptor is probably not associated with G-proteins, but may have intramolecular-free sulfhydryl groups. The specific activity of the isolated NPY-Y2 receptor is 15.8 nmol/mg of protein. The isolated receptor retained its capacity to bind to 125I-NPY, specific to NPY and peptide YY, and showed no cross-reactivity with any other peptides. Highly purified (10(9)-fold purification) NPY receptor from the brain was identified as the Y2 subtype as demonstrated by its affinity to C-terminal fragments of NPY, including NPY-(13-36).
Wimalawansa
S J
SJ
Department of Internal Medicine, University of Texas Medical Branch, Galveston 77555-1065, USA.
D000818:Animals; D001923:Brain Chemistry; D003432:Cross-Linking Reagents; D003902:Detergents; D008562:Membrane Glycoproteins / Q000302:isolation & purification* / Q000378:metabolism; D008970:Molecular Weight; D009478:Neuropeptide Y / Q000031:analogs & derivatives / Q000378:metabolism*; D010446:Peptide Fragments / Q000378:metabolism; D011485:Protein Binding; D011487:Protein Conformation; D017476:Receptors, Neuropeptide Y / Q000302:isolation & purification / Q000378:metabolism*; D012995:Solubility; D013552:Swine
D003432:Cross-Linking Reagents; D003902:Detergents; D008562:Membrane Glycoproteins; D009478:Neuropeptide Y; D010446:Peptide Fragments; D017476:Receptors, Neuropeptide Y