Biotinylated endothelin as a probe for the endothelin receptor.
Biotinylated derivatives of endothelin (ET)-1 were prepared by chemical modification of ET-1 with sulfosuccinimidyl 6-(biotinamido) hexanoate. Two major biotinylated ET analogs were purified by reversed-phase high performance liquid chromatography. Edman degradation indicated that the first eluting peptide contains one biotin residue on lysine at position 9, while the second derivative contains an additional biotin residue at position 1. Competition binding studies to mouse osteoblastic cell line MC3T3-E1 using 125I-labeled ET-1 revealed IC50 values of 5, 30 and 600 nM for native ET, the mono- and the dibiotinylated ET analog, respectively. A similar order of potency was obtained when these ET derivatives were examined for stimulation of DNA synthesis in MC3T3-E1 cells. In addition, incubation of MC3T3-E1 cells with the monobiotinylated ET and subsequent addition of rhodamine-avidin resulted in an evenly distributed fluorescence over the cell surface. The fluorescence observed was completely abolished in the presence of an excess of native ET. Thus the monobiotinylated ET proves to be useful for localization of the ET receptors.
Schvartz
I
I
Division of Biology, Glaxo Research Institute, Research Triangle Park, NC 27709.
Gitlin
G
G
Amarant
T
T
Ittoop
O
O
Hazum
E
E
D000595:Amino Acid Sequence; D000818:Animals; D001710:Biotin / Q000031:analogs & derivatives* / Q000378:metabolism*; D002460:Cell Line; D016232:Endothelins / Q000737:chemistry / Q000378:metabolism*; D006651:Histocytochemistry; D008856:Microscopy, Fluorescence; D015335:Molecular Probes; D008969:Molecular Sequence Data; D010006:Osteoblasts / Q000378:metabolism; D011956:Receptors, Cell Surface / Q000378:metabolism*; D017466:Receptors, Endothelin
D016232:Endothelins; D015335:Molecular Probes; D011956:Receptors, Cell Surface; D017466:Receptors, Endothelin; D001710:Biotin