Endocrinology, Vol 126, 3218-3222, Copyright © 1990 by Endocrine Society

ARTICLES

Bovine cerebellum endothelin receptor: solubilization and identification

I Schvartz, O Ittoop and E Hazum
Division of Endocrinology, Glaxo Research Laboratories, Research Triangle Park, North Carolina 27709.

Endothelin receptors were solubilized from bovine cerebellum membrane preparations in an active form by using the zwitterionic detergent CHAPS, [3-(3-cholamidopropyl)dimethylammonio]-1-propane sulfonic acid. The solubilized receptors displayed high affinity, saturability, and specificity. The dissociation constant (Kd) for endothelin was 7 +/- 2 nM, and the number of binding sites was 600 +/- 200 fmol/mg protein. These results are similar to those obtained for the membrane-bound receptor and suggest that during solubilization the binding characteristics of the receptor are preserved. Attempts to purify the solubilized receptors in an active form using affinity chromatography techniques, i.e. Affi-gel 15 column coupled to endothelin, were not successful. Nevertheless, identification of the receptors was achieved by affinity chromatography of the solubilized proteins and subsequent iodination. Autoradiographic analysis of sodium dodecyl sulfate- polyacrylamide gel electrophoresis revealed a major protein with an apparent mol wt of 50 kD. Taken together with our previous findings, this result suggests that the 50-kD band represents the endothelin receptor.