Discriminative affinity labelling of opioid receptors by enkephalin and morphiceptin analogues containing 3-nitro-2-pyridinesulphenyl-activated thiol residues.
The thiol groups of leucinthiol, cysteamine and cysteine incorporated into opioid peptides enkephalin and morphiceptin were activated by the 3-nitro-2-pyridinesulphenyl (Npys) group to form mixed disulphides highly reactive to a free thiol. Enkephalin analogues containing Npys-leucinthiol or -cysteine at positions 4, 5 and 6 exhibited high affinities for both mu and delta receptors, while morphiceptin analogues containing Npys-cysteine at positions 4 and 5 showed relatively weak affinity only for mu receptors. When these S-activated opioid peptides were incubated with rat brain membrane preparations, it was found, by binding assay using radiolabelled and non-labelled [D-Ala2,MePhe4,Gly-ol5]enkephalin, that they label mu opioid receptors in a dose-dependent manner. The concentrations required to label half of the receptors were 0.2-2 microM for enkephalins and 10-30 microM for morphiceptins. These results suggested that the thiol group labelled by S-activated enkephalins and morphiceptins is present in the ligand binding site of receptor protein, but not in GTPase-binding protein.
Shimohigashi
Y
Y
Department of Chemistry, Faculty of Science, Kyushu University, Fukuoka, Japan.
Takada
K
K
Sakamoto
H
H
Matsumoto
H
H
Yasunaga
T
T
Kondo
M
M
Ohno
M
M
D000345:Affinity Labels; D000595:Amino Acid Sequence; D004723:Endorphins / Q000737:chemistry*; D020875:Enkephalin, Ala(2)-MePhe(4)-Gly(5)-; D016308:Enkephalin, Leucine-2-Alanine / Q000032:analysis; D004745:Enkephalins / Q000032:analysis / Q000737:chemistry*; D007202:Indicators and Reagents; D008969:Molecular Sequence Data; D010455:Peptides / Q000032:analysis; D011725:Pyridines; D011869:Radioligand Assay; D011957:Receptors, Opioid / Q000032:analysis*
D000345:Affinity Labels; D004723:Endorphins; D004745:Enkephalins; D007202:Indicators and Reagents; D010455:Peptides; D011725:Pyridines; D011957:Receptors, Opioid; D020875:Enkephalin, Ala(2)-MePhe(4)-Gly(5)-; D016308:Enkephalin, Leucine-2-Alanine; C065586:3-nitro-2-pyridinesulfenyl; C028889:morphiceptin