Affinity labeling of the mu opioid receptor in bovine striatal membranes with [3H]-14 beta-(bromoacetamido)-7,8-dihydromorphine.
[3H]-14 beta-(Bromoacetamido)-7,8-dihydromorphine ([3H]H2BAM) was synthesized and tested for its ability to selectively label mu opioid receptors in bovine striatal membranes. Incubating membranes with N-tosyl-L-phenylalanine chloromethyl ketone and dithiothreitol before the addition of [3H]H2BAM reduced nonspecific [3H]H2BAM binding so that [3H]H2BAM binding to opioid receptors was up to 70% of the total [3H]H2BAM binding and was dependent on [3H]H2BAM concentration, incubation time, and pH of the reaction. At pH 7.5, [3H]H2BAM bound selectively to the mu opioid receptor, but mainly noncovalently. After the initial binding of [3H]H2BAM 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 [3H]H2BAM associated with the receptor becoming covalently bound to the opioid receptor. The mu-selective peptide [D-Ala2,(Me)Phe4,Gly(ol)5]enkephalin inhibited [3H]H2BAM labeling of membranes, while delta- or kappa-selective compounds were ineffective. Both NaCl and the nonhydrolyzable guanine nucleotide analog guanylyl 5'-imidodiphosphate reduced the incorporation of [3H]H2BAM into membranes. When [3H]H2BAM-labeled striatal membranes were separated under reducing conditions on a sodium dodecyl sulfate-polyacrylamide gel, two proteins with molecular weights of 54,000 and 44,000 were specifically labeled. The 54-kDa protein was present in a greater amount than the 44-kDa protein. Both proteins bound to wheat germ agglutinin-Sepharose and concanavalin A-Sepharose, suggesting that both proteins contain multiple carbohydrate moieties. Despite the inclusion of protease inhibitors, the 44-kDa protein may be a proteolytic fragment of the 54-kDa protein.(ABSTRACT TRUNCATED AT 250 WORDS)
Bidlack
J M
JM
Department of Pharmacology, University of Rochester School of Medicine and Dentistry, New York 14642.
Kaplan
R A
RA
Subbramanian
R A
RA
Seyed-Mozaffari
A
A
Archer
S
S
D000345:Affinity Labels / Q000378:metabolism; D000478:Alkylation; D000700:Analgesics / Q000378:metabolism*; D000818:Animals; D001667:Binding, Competitive; D002417:Cattle; D002462:Cell Membrane / Q000187:drug effects / Q000378:metabolism; D002846:Chromatography, Affinity; D003208:Concanavalin A; D003342:Corpus Striatum / Q000378:metabolism*; D006165:Guanylyl Imidodiphosphate / Q000494:pharmacology; D004091:Hydromorphone / Q000031:analogs & derivatives* / Q000138:chemical synthesis / Q000378:metabolism; D007700:Kinetics; D015394:Molecular Structure; D009294:Narcotics / Q000494:pharmacology; D017450:Receptors, Opioid, mu / Q000302:isolation & purification / Q000378:metabolism*; D012685:Sepharose / Q000031:analogs & derivatives; D012965:Sodium Chloride / Q000494:pharmacology; D014316:Tritium
D000345:Affinity Labels; D000700:Analgesics; D009294:Narcotics; D017450:Receptors, Opioid, mu; C042758:wheat germ agglutinin-sepharose 6MB; D014316:Tritium; D003208:Concanavalin A; C076028:14-bromoacetamido-7,8-dihydromorphinone; D006165:Guanylyl Imidodiphosphate; D012965:Sodium Chloride; D012685:Sepharose; D004091:Hydromorphone