Biotinylation of a bombesin/gastrin-releasing peptide analogue for use as a receptor probe.
The development of a biotinylated bombesin/gastrin-releasing peptide (GRP) for use as a receptor probe is reported. The lysine13 of a GRP-27 was substituted by arginine and lysine was added to the amino terminus. Biotinylation of the N-terminal lysine was performed. The biotinylated peptide was purified by HPLC and characterized by mass spectral analysis. Binding studies with murine Swiss 3T3 fibroblasts, cells known to express bombesin/GRP receptors, yielded a dissociation curve for the biotinylated GRP-27 analogue (biotin-Lysyl[Asp12,Arg13]GRP-27) which was nearly identical to that of native GRP. Using studies of gastrin release from isolated canine G cells, equipotent functional activity of the biotinylated probe and unmodified GRP was demonstrated. Measurements of retained 125I-avidin confirmed that the biotin/avidin interaction could occur once the biotin-peptide complex was bound. Applicability of the probe was demonstrated with fluorescent microscopy using avidin-FITC on Swiss 3T3 fibroblasts. In conclusion, a novel biotinylated bombesin/GRP analogue has been developed which retains the functional characteristics of the native peptide and is a useful probe for receptor studies.
Anton
P A
PA
Department of Medicine, UCLA.
Reeve
J R
JR
Rivier
J E
JE
Vidrich
A
A
Schepp
W
W
Shanahan
F
F
D000595:Amino Acid Sequence; D000818:Animals; D001710:Biotin / Q000031:analogs & derivatives* / Q000737:chemistry / Q000378:metabolism; D001839:Bombesin / Q000737:chemistry / Q000378:metabolism*; D002460:Cell Line; D019886:Gastrin-Releasing Peptide; D006801:Humans; D008856:Microscopy, Fluorescence; D015335:Molecular Probes; D008969:Molecular Sequence Data; D010455:Peptides / Q000737:chemistry / Q000378:metabolism*; D018004:Receptors, Bombesin; D017981:Receptors, Neurotransmitter / Q000378:metabolism*
D015335:Molecular Probes; D010455:Peptides; D018004:Receptors, Bombesin; D017981:Receptors, Neurotransmitter; C069745:paotin-lysyl-GRP-27, Asp(12),Arg(13)-; D001710:Biotin; D019886:Gastrin-Releasing Peptide; D001839:Bombesin