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
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
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.