Jm. Bidlack et al., AFFINITY LABELING OF THE MU-OPIOID RECEPTOR IN BOVINE STRIATAL MEMBRANES WITH [H-3] 14-BETA-(BROMOACETAMIDO)-7,8-DIHYDROMORPHINE, Biochemistry, 32(26), 1993, pp. 6703-6711
[H-3]-14beta-(Bromoacetamido)-7,8-dihydromorphine ([H-3]H-2BAM) was sy
nthesized and tested for its ability to selectively label mu opioid re
ceptors in bovine striatal membranes. Incubating membranes with N-tosy
l-L-phenylalanine chloromethyl ketone and dithiothreitol before the ad
dition of [H-3]H-2BAM reduced nonspecific [H-3]H-2BAM binding so that
[H-3]H-2BAM binding to opioid receptors was up to 70% of the total [H-
3]H-2BAM binding and was dependent on [H-3]H-2BAM concentration, incub
ation time, and pH of the reaction. At pH 7.5, [H-3]H-2BAM bound selec
tively to the mu opioid receptor, but mainly noncovalently. After the
initial binding of [H-3]H-2BAM to the receptor, membranes were washed
and then incubated at 37-degrees-C in 50 mM Tris-HCl, pH 8.5, for 3 h,
a time that resulted in greater than 80% of the [H-3]H-2BAM associate
d with the receptor becoming covalently bound to the opioid receptor.
The mu-selective peptide [D-Ala2,(Me)Phe4,Gly(ol)5]enkephalin inhibite
d [H-3]H-2BAM labeling of membranes, while delta- or kappa-selective c
ompounds were ineffective. Both NaCl and the nonhydrolyzable guanine n
ucleotide analog guanylyl 5'-imidodiphosphate reduced the incorporatio
n of [H-3]H-2BAM into membranes. When [H-3]H-2-BAM-labeled striatal me
mbranes were separated under reducing conditions on a sodium dodecyl s
ulfate-polyacrylamide gel, two proteins with molecular weights of 54 0
00 and 44 000 were specifically labeled. The 54-kDa protein was presen
t in a greater amount than the 44-kDa protein. Both proteins bound to
wheat germ agglutinin-Sepharose and concanavalin A-Sepharose, suggesti
ng that both proteins contain multiple carbohydrate moieties. Despite
the inclusion of protease inhibitors, the 44-kDa protein may be a prot
eolytic fragment of the 54-kDa protein. Alternatively, these two prote
ins may be either different types of mu opioid receptors, or different
degrees of posttranslational modification may account for the differe
nce in the apparent molecular weight of the two proteins.