Purification and characterization of bovine lung endothelin receptor.
Endothelin receptor was purified from bovine lung by a rapid and simple two-step procedure: 1) solubilization with the detergent 3[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate and digitonin and 2) affinity chromatography using biotinylated endothelin and avidin-agarose. Starting from 3.5 kg of bovine lung, about 200 micrograms of pure receptor were obtained. Microsequencing of tryptic fragments of the purified protein revealed a high sequence similarity with the rat endothelin ETB receptor that has very recently been cloned by expression cloning and shown to be nonselective in terms of the ligand specificity. Purification of the receptor in the presence of low (1 mM) and high (50 mM) concentrations of EDTA yielded, as a major form, 34- and 52-kDa species, respectively, indicating that the lower Mr species (34 kDa) is a proteolytic product of the 52-kDa species. Interestingly, this metal proteinase-mediated limited proteolysis did not affect the ligand binding properties of the receptor.
Kozuka
M
M
Department of Biological Sciences, Tokyo Institute of Technology, Japan.
Ito
T
T
Hirose
S
S
Lodhi
K M
KM
Hagiwara
H
H
D000595:Amino Acid Sequence; D000818:Animals; D001667:Binding, Competitive; D001710:Biotin / Q000378:metabolism; D002417:Cattle; D002846:Chromatography, Affinity; D002851:Chromatography, High Pressure Liquid; D004591:Electrophoresis, Polyacrylamide Gel; D016232:Endothelins / Q000378:metabolism*; D008024:Ligands; D008168:Lung / Q000378:metabolism*; D008969:Molecular Sequence Data; D008970:Molecular Weight; D011956:Receptors, Cell Surface / Q000302:isolation & purification / Q000378:metabolism*; D017466:Receptors, Endothelin
D016232:Endothelins; D008024:Ligands; D011956:Receptors, Cell Surface; D017466:Receptors, Endothelin; D001710:Biotin