125I-neuropeptide Y binding activity of pig spleen cell membranes: effect of solubilisation.
Crude preparations of pig spleen cell membranes were obtained by differential centrifugation. 125I-NPY bound specifically to these membranes with a KD of 56 +/- 13 pM and Bmax of 44 +/- 4.0 fmols/mg protein. After treatment with 1% CHAPS* and 10 mM 2-mercaptoethanol in the presence of 2 microM leupeptin, 2 microM pepstatin A, 10 microM phosphoramidon, 200 microM PMSF and 0.1% bacitracin, followed by centrifugation at 100,000 g a soluble preparation was obtained that contained a single population of specific 125I-NPY binding sites. The KD of the soluble receptor was significantly higher at 1.38 +/- 0.2 nM (P less than 0.01) but the Bmax of 59.6 +/- 6.6 fmols/mg protein was similar (N.S.). This is the first description of a method for obtaining NPY receptors in soluble form and should enable their purification and characterisation, though the low affinity of the soluble receptor may reflect disruption of the ligand binding site upon removal of the receptor from the membrane.
Clinical Pharmacology Unit, University of Cambridge Clinical School, Addenbrooke's Hospital, U.K.
J S
JS
Price
M J
MJ
Brown
D000818:Animals; D009320:Atrial Natriuretic Factor / Q000378:metabolism; D002462:Cell Membrane / Q000737:chemistry* / Q000378:metabolism; D008024:Ligands; D015288:Neurokinin A / Q000378:metabolism; D009478:Neuropeptide Y / Q000378:metabolism*; D017476:Receptors, Neuropeptide Y; D017981:Receptors, Neurotransmitter / Q000302:isolation & purification* / Q000378:metabolism; D012995:Solubility; D013154:Spleen / Q000166:cytology / Q000378:metabolism*; D013373:Substance P / Q000378:metabolism; D013552:Swine; D014660:Vasoactive Intestinal Peptide / Q000378:metabolism
D008024:Ligands; D009478:Neuropeptide Y; D017476:Receptors, Neuropeptide Y; D017981:Receptors, Neurotransmitter; D013373:Substance P; D014660:Vasoactive Intestinal Peptide; D009320:Atrial Natriuretic Factor; D015288:Neurokinin A