32P-labeled opioid peptides with high affinity for the delta-opioid receptor.
We describe a mild and convenient labeling method for obtaining opioid radioligands which exhibit high specific activity together with a high affinity for the delta-opioid receptor. We chemically synthesized and tested the affinity of enkephalin- and deltorphin-like peptides that contain a phosphorylation site at their C-terminus. The peptide YdAGFLTPRRASLGC (peptide B), labeled to 700 Ci/mmol in the presence of cAMP-dependent protein kinase and [gamma-32P]ATP, bound to the receptor with high affinity (Kd = 3.62 +/- 0.29 nM). This peptide was also chemically coupled to bovine serum albumin and provided a multivalent opioid protein (B-BSA) with interesting properties: compared with peptide B, B-BSA was a better substrate for the kinase (100% 32P incorporation, sp act > or = 7000 Ci/mmol when labeled) and a better ligand for the receptor (Kd = 0.20 +/- 0.02 nM). The concept of peptide extension by a short phosphorylatable sequence should be more generally applicable to other small peptidic hormones or neurotransmitters and provide useful probes for biochemical studies and expression cloning of membrane receptors.
Kieffer
B L
BL
Ecole Supérieure de Biotechnologie, Strasbourg, France.
Black
D
D
Hirth
C G
CG
D000595:Amino Acid Sequence; D000818:Animals; D017868:Cyclic AMP-Dependent Protein Kinases / Q000378:metabolism; D004723:Endorphins / Q000378:metabolism* / Q000494:pharmacology; D007700:Kinetics; D008969:Molecular Sequence Data; D010761:Phosphorus Radioisotopes; D010766:Phosphorylation; D011485:Protein Binding; D017465:Receptors, Opioid, delta / Q000378:metabolism*; D012680:Sensitivity and Specificity; D012710:Serum Albumin, Bovine / Q000378:metabolism
D004723:Endorphins; D010761:Phosphorus Radioisotopes; D017465:Receptors, Opioid, delta; D012710:Serum Albumin, Bovine; D017868:Cyclic AMP-Dependent Protein Kinases