title: Biotin derivatives of endothelin: utilization for affinity purification of endothelin receptor.
abstract: Three different types of biotinylated endothelin 1 (ET-1) derivatives, [Cys1]-biotinylated ET-1, [Lys9]-biotinylated ET-1, and [Cys1][Lys9]-dibiotinylated ET-1, were obtained when the biotinylation reaction was carried out with sulfosuccinimidyl-6-(biotinamido)hexanoate in an aqueous solvent. The binding of [Lys9]-biotinylated ET-1 to the ET receptor was as efficient as that of natural ET-1, whereas the binding of either [Cys1]-biotinylated ET-1 or [Cys1][Lys9]-dibiotinylated ET-1 was significantly reduced. When ET-1 was reacted with succinimidyl-6-(biotinamido)hexanoate in an organic solvent, ET-1 was exclusively modified at lysine 9. The ET receptor was then isolated from human placenta by affinity chromatography with [Lys9]-biotinylated ET-1 and avidin-agarose. The purified ET receptor was active in ET binding and was resolved by sodium dodecyl sulfate-polyacrylamide gel electrophoresis into two polypeptides with apparent molecular masses of 45 and 35 kDa. The NH2-terminal amino acid sequence indicated that the two polypeptides were from an identical subtype of the ET receptor (ETB, the ligand-nonselective type). A signal peptide from Met1 to Gly26 was missing from the 45-kDa ETB, whereas 64 amino acids at the NH2 terminus were missing from the 35-kDa ETB due to proteolytic cleavage which occurred between Arg64 and Ser65. Indeed, incubation of purified ETB with endopeptidase Arg-C resulted in degradation of the 45-kDa ETB, giving rise to the 35-kDa species by a specific cleavage at Arg64. The 35-kDa ETB was active in binding to ET-1, indicating that the NH2-terminal 64-amino-acid residues are not essential for ligand binding.(ABSTRACT TRUNCATED AT 250 WORDS)
authors.affiliation: Department of Molecular Genetics, Nippon Roche Research Center, Kanagawa, Japan.
authors.forename: N
authors.initials: N
authors.lastname: Akiyama
authors.forename: O
authors.initials: O
authors.lastname: Hiraoka
authors.forename: Y
authors.initials: Y
authors.lastname: Fujii
authors.forename: H
authors.initials: H
authors.lastname: Terashima
authors.forename: M
authors.initials: M
authors.lastname: Satoh
authors.forename: K
authors.initials: K
authors.lastname: Wada
authors.forename: Y
authors.initials: Y
authors.lastname: Furuichi
mesh_terms: D000595:Amino Acid Sequence; D001360:Avidin / Q000737:chemistry; D001710:Biotin / Q000031:analogs & derivatives* / Q000737:chemistry; D002846:Chromatography, Affinity / Q000379:methods; D002847:Chromatography, Agarose / Q000379:methods; D010450:Endopeptidases; D016232:Endothelins / Q000737:chemistry*; D005260:Female; D006801:Humans; D006868:Hydrolysis; D008969:Molecular Sequence Data; D011247:Pregnancy; D011257:Pregnancy Proteins / Q000302:isolation & purification; D017466:Receptors, Endothelin / Q000187:drug effects / Q000302:isolation & purification*; D012697:Serine Endopeptidases / Q000494:pharmacology; D013388:Succinimides
chemical_list: D016232:Endothelins; D011257:Pregnancy Proteins; D017466:Receptors, Endothelin; D013388:Succinimides; C061443:sulfosuccinimidyl 6-(biotinamido)hexanoate; D001360:Avidin; D001710:Biotin; D010450:Endopeptidases; C044499:submandibular proteinase A; D012697:Serine Endopeptidases