Biotinyl C-terminal-extended motilin as a biologically active receptor probe.
The synthesis, purification, and characterization of biotinylated analogues of motilin are reported. The C-terminal of canine motilin was extended by the addition of a cysteine residue, and then biotinylated. Biotinyl motilin was purified by following HPLC and characterized by amino acid analysis. Biotinylation of the ligand was confirmed by ELISA assay with the avidin-biotin system. Biotinyl motilin showed similar affinity for binding to rabbit gastric membrane fraction compared to unlabeled canine motilin, and also retained functional activity in its ability to cause contraction of rabbit duodenal segments. To determine the binding of biotinyl motilin in isolated rabbit antral smooth muscle, cells were incubated with the biotinyl motilin with and without excess of unlabeled motilin. Subsequent addition of avidin-biotinylated peroxidase complex showed the distribution of reaction products over the cell surface. Bioactive biotinyl motilin provides a useful probe for the demonstration of cell surface motilin receptors and will facilitate receptor purification and characterization.
Sakai
T
T
Gastrointestinal Research Laboratory, Gunma University, Maebashi, Japan.
Satoh
M
M
Hayashi
H
H
Fujikura
K
K
Sano
I
I
Koyama
H
H
Tatemoto
K
K
Itoh
Z
Z
D000595:Amino Acid Sequence; D000818:Animals; D001710:Biotin / Q000031:analogs & derivatives* / Q000138:chemical synthesis / Q000378:metabolism; D004285:Dogs; D005748:Gastric Fundus / Q000166:cytology / Q000378:metabolism*; D006651:Histocytochemistry / Q000379:methods*; D015335:Molecular Probes; D008969:Molecular Sequence Data; D009037:Motilin / Q000031:analogs & derivatives* / Q000138:chemical synthesis / Q000378:metabolism*; D009130:Muscle, Smooth / Q000378:metabolism*; D011817:Rabbits; D011964:Receptors, Gastrointestinal Hormone / Q000302:isolation & purification*; D018013:Receptors, Neuropeptide / Q000302:isolation & purification*
D015335:Molecular Probes; D011964:Receptors, Gastrointestinal Hormone; D018013:Receptors, Neuropeptide; C087835:biotinyl(Cys(23))motilin; C050802:motilin receptor; D009037:Motilin; D001710:Biotin