PURIFICATION AND CHARACTERIZATION OF B-2 BRADYKININ RECEPTOR FROM RAT UTERUS

A B-2 bradykinin (BK) receptor was solubilised and partially purified from rat uterine membranes by a combination of ammonium sulphate precipitation, desalting on Sephadex G-50, and hydroxyapatite and wheat germ agglutinin affinity chromatography. The partially purified BK receptor, enriched 1,500-fold, was then cross-linked to I-125-Tyr(0)-BK using disuccinimidyl suberate and purified to homogeneity as a single protein species on two-dimensional gel electrophoresis with a molecular mass of 81 kDa. This molecular size was in agreement with the value of 80-120 kDa estimated from Sephacryl 300 size exclusion column chromatography of the B-2 receptor. The partially purified and the crude solubilised B-2 BK receptor from rat uterus showed similar affinities for BK and the BK analogues iodo-Tyr(0)-BK, D-Phe(7)-BK, and des-Arg(9)-BK, indicating that the ligand binding specificity of the receptor had been retained during the purification procedures. The biochemical properties of the solubilised B-2 BK receptor correspond to those of a hydrophobic acidic glycoprotein (isoelectric focusing gave a value of 4.5-4.7) that binds specifically to wheat germ agglutinin but has no affinity for either concanavalin A or lentil lectin, suggesting the absence of terminal mannose or glucose residues.