Affinity cross-linking of bradykinin B2 receptors in guinea pig ileum membranes.

Affinity cross-linking of the bradykinin B2 receptor was performed using [125I-Tyr8]bradykinin, disulfosuccinimidyl tartrate as linker and crude membranes from guinea pig ileum smooth muscle immobilized on Whatman GF/B glass fiber filters. After SDS (sodium dodecylsulfate)-polyacrylamide gel electrophoresis under reducing conditions one major band of an apparent molecular mass of 66 kDa was obtained. The labeling intensity of this band was diminished in the presence of both unlabeled bradykinin and the bradykinin B2 receptor selective antagonist Hoe 140 ([D-Arg0,Hyp3,Thi5,D-Tic7,Oic8]bradykinin) but not by the bradykinin B1 receptor agonist des-Arg9-bradykinin. Under non-reducing conditions this band shifted to a 59 kDa position. Considering the molecular mass of covalently bound [125I-Tyr8]bradykinin (1.2 kDa) the bradykinin B2 receptor in guinea pig ileum smooth muscle membranes is a 65 kDa protein. Its structure seems to be very similar to, and yet slightly distinct from, the 69 kDa bradykinin B2 receptor in human fibroblasts recently described (Abd Alla et al., 1993, J. Biol. Chem. 268, 17277) suggesting putative differences between bradykinin B2 receptors in various tissues.