Biotin derivatives of endothelin: utilization for affinity purification of endothelin receptor.
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)
Akiyama
N
N
Department of Molecular Genetics, Nippon Roche Research Center, Kanagawa, Japan.
Hiraoka
O
O
Fujii
Y
Y
Terashima
H
H
Satoh
M
M
Wada
K
K
Furuichi
Y
Y
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
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