title: Separation and purification of 34- and 52-kDa species of bovine lung endothelin receptors and identification of the 34-kDa species as a degradation product.
abstract: We solubilized bovine lung endothelin receptors in a nonaggregated state and identified 34- and 52-kDa species of endothelin receptor by affinity labeling. The ratio of 34-kDa species to 52-kDa species varied widely from sample to sample, suggesting that the 34-kDa species is a proteolytic product; we therefore examined the effect of various proteinase inhibitors and found that EDTA (greater than 50 mM) was very effective in preventing the conversion. These results indicate that the lower Mr species arose from the 52-kDa receptor as a result of limited proteolysis by metal proteinase(s). The resulting 34-kDa species had endothelin-binding activity, was very stable, and was not processed further even in the absence of proteinase inhibitors. We therefore first tried to purify the 34-kDa species by affinity chromatography. Bovine lung extracts were mixed with biotinylated endothelin and the ligand-receptor complexes formed were isolated with avidin-agarose. Starting from 3.5 kg of bovine lung, approximately 200 micrograms of the pure receptor was obtained. Microsequencing of two well-separated tryptic fragments yielded the following partial amino acid sequences: Ala-Asn-Asp-His-Gly-Tyr-Asp-Asn-Phe and Ser-Gly-Met-Gln-Ile-Ala-Leu-Asn-Asp-His-Leu-Lys.
authors.affiliation: Department of Biological Sciences, Tokyo Institute of Technology, Japan.
authors.forename: H
authors.initials: H
authors.lastname: Hagiwara
authors.forename: M
authors.initials: M
authors.lastname: Kozuka
authors.forename: H
authors.initials: H
authors.lastname: Sakaguchi
authors.forename: S
authors.initials: S
authors.lastname: Eguchi
authors.forename: T
authors.initials: T
authors.lastname: Ito
authors.forename: S
authors.initials: S
authors.lastname: Hirose
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D002417:Cattle; D004492:Edetic Acid / Q000494:pharmacology; D008168:Lung / Q000737:chemistry*; D008969:Molecular Sequence Data; D008970:Molecular Weight; D011956:Receptors, Cell Surface / Q000302:isolation & purification* / Q000378:metabolism; D017466:Receptors, Endothelin
chemical_list: D011956:Receptors, Cell Surface; D017466:Receptors, Endothelin; D004492:Edetic Acid