Reversible affinity labeling of opioid receptors via disulfide bonding: discriminative labeling of mu and delta subtypes by chemically activated thiol-containing enkephalin analogs.
The 3-nitro-2-pyridinesulfenyl (Npys) group bound to a mercapto group is a highly activated electrophilic reagent, which only reacts with a free mercapto group to form a disulfide bond via the thiol-disulfide exchange reaction. We incorporated the Npys group into enkephalin analogs to affinity label mu and delta opioid receptors. When rat brain membranes were incubated with [D-Ala2,Leu(CH2SNpys)5]enkephalin, and assayed for the inhibition of binding of DAGO and DSLET enkephalin analogs to opioid receptors, the number of receptors decreased sharply, depending upon the concentration of this SNpys-containing enkephalin. It was found that this enkephalin analog occupies mu receptors highly specifically (EC50 = 51 nM) and almost 100 times more selectively than delta receptors. In contrast, [D-Ala2,Leu5]enkephalyl-Cys(Npys)6 attached covalently to delta receptors (EC50 = 34 nM) about 150 times more selectively than to mu receptors. Although N-ethylmaleimide also inhibited the binding of DAGO and DSLET, four to six orders of magnitude higher concentrations were required as compared to SNpys-containing enkephalins. When enkephalin-bound rat membranes were treated with dithiothreitol, the loss of receptors was reversed, depending upon the concentration of and incubation time with dithiothreitol. The recovery was much faster (about 1,000 times) for delta receptors than for mu receptors. The present results indicated that both mu and delta receptors in rat brain consist of a free mercapto group near the enkephalin binding site and that SNpys-containing enkephalins can label these mercapto groups discriminatively. The disulfide bond between [D-Ala2,Leu5]enkephalyl-Cys6 and delta receptors appears to be exposed, while that between [D-Ala2,Leu(CH2-SNpys)5] enkephalin and mu receptors is shielded.
Manufacturing Process Development Division, Otsuka Pharmaceutical Co., Ltd., Saga Factory.
T
T
Yasunaga
S
S
Motoyama
T
T
Nose
H
H
Kodama
M
M
Kondo
Y
Y
Shimohigashi
D000345:Affinity Labels / Q000737:chemistry / Q000378:metabolism; D000818:Animals; D001665:Binding Sites; D001921:Brain / Q000378:metabolism; D004220:Disulfides / Q000737:chemistry; D004229:Dithiothreitol; D020875:Enkephalin, Ala(2)-MePhe(4)-Gly(5)-; D004743:Enkephalin, Leucine / Q000031:analogs & derivatives / Q000378:metabolism; D004745:Enkephalins / Q000737:chemistry* / Q000378:metabolism*; D005033:Ethylmaleimide; D066298:In Vitro Techniques; D007700:Kinetics; D015394:Molecular Structure; D051381:Rats; D017465:Receptors, Opioid, delta / Q000737:chemistry / Q000378:metabolism*; D017450:Receptors, Opioid, mu / Q000737:chemistry / Q000378:metabolism*; D013439:Sulfhydryl Reagents
D000345:Affinity Labels; D004220:Disulfides; D004745:Enkephalins; D017465:Receptors, Opioid, delta; D017450:Receptors, Opioid, mu; D013439:Sulfhydryl Reagents; D020875:Enkephalin, Ala(2)-MePhe(4)-Gly(5)-; D004743:Enkephalin, Leucine; C034318:enkephalin, Ser(2), Leu(5), Thr(6)-; D005033:Ethylmaleimide; D004229:Dithiothreitol