SEPARATION AND PURIFICATION OF 34-KDA AND 52-KDA SPECIES OF BOVINE LUNG ENDOTHELIN RECEPTORS AND IDENTIFICATION OF THE 34-KDA SPECIES AS A DEGRADATION PRODUCT

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 (> 50 mM) was very effective in preventing the conversion. These results indicate that the lower M(r) 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-mu-g 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.