ANTISERA AGAINST PEPTIDES DERIVED FROM A PURIFIED MU-OPIOID BINDING-PROTEIN RECOGNIZE THE PROTEIN AS WELL AS MU-OPIOID RECEPTORS IN BRAIN-REGIONS AND A CELL-LINE

Citation
Tl. Gioannini et al., ANTISERA AGAINST PEPTIDES DERIVED FROM A PURIFIED MU-OPIOID BINDING-PROTEIN RECOGNIZE THE PROTEIN AS WELL AS MU-OPIOID RECEPTORS IN BRAIN-REGIONS AND A CELL-LINE, Molecular pharmacology, 44(4), 1993, pp. 796-801
Citations number
24
Categorie Soggetti
Pharmacology & Pharmacy",Biology
Journal title
ISSN journal
0026895X
Volume
44
Issue
4
Year of publication
1993
Pages
796 - 801
Database
ISI
SICI code
0026-895X(1993)44:4<796:AAPDFA>2.0.ZU;2-5
Abstract
Two peptides, which have no significant homology with known protein st ructures, were obtained by microsequencing of a mu-opioid binding prot ein purified to homogeneity from bovine striatal membranes. Polyclonal antibodies generated against portions of these peptides immunoprecipi tated up to 65% of radiolabeled purified opioid binding protein. Seque ntial immunoprecipitations, using antibodies directed against portions of the two different peptides, confirmed that the peptides are derive d from the same protein. Immunoblots of the protein with antipeptide a ntibodies revealed a protein band corresponding to the molecular weigh t of denatured reduced mu-opioid binding protein. The immunoresponse w as blocked by the appropriate peptide and was not observed with irrele vant antisera. The antipeptide antibodies were used for immunoblots of sodium dodecyl sulfate extracts of tissues from bovine brain regions and of the mu receptor-containing cell line SK-N-SH. Affinity-purified antipeptide antibody detected an immunoreactive protein of molecular weight 65,000 in brain regions containing high levels of mu-opioid rec eptors (striatum, thalamus, hippocampus, and frontal cortex) and in th e cell line SK-N-SH. Pons, which contains low levels of receptors, pro duced a a barely detectable signal, whereas white matter, HeLa cells, and C6 glioma cells, devoid of opioid binding activity, produced no de tectable signal. The correlation between immunoreactivity and the pres ence of mu-opioid binding in brain regions and cell lines and the corr espondence of the molecular weight of the immunoreactive protein to th at of mu-opioid receptors provide strong evidence that the peptide ant isera recognize mu receptors.