Characterization of soluble bradykinin receptor-like binding sites

Bradykinin (BK) receptor-like binding sites were solubilized from a particulate fraction of bovine uterine myometrium (BUM) using the zwitterionic detergent 3-[(3-cholamidopropyl)-dimethylammonio]-1-propane sulfonate (CHAPS). Scatchard analysis of [125I-Tyr1]kallidin (T1K) binding revealed a single class of soluble binding sites with KD = 0.35 nM and Bmax = 0.13 pmol/mg protein. The soluble binding sites exhibited a kinin-binding specificity comparable to that of the particulate BUM receptor-like sites. Soluble T1K binding association kinetics were first-order at the four T1K concentrations examined. A plot of the pseudo-first order rate constant (Kobs) versus T1K concentration was linear, and values for the association (k1) and dissociation (k-1) rate constants were obtained. These rate constants yielded a kinetically derived equilibrium dissociation constant (KD = 0.64 nM) which was comparable to that obtained by Scatchard analysis. Biphasic dissociation of bound T1K was resolved into rapid and slow dissociation phases. The rate constant (k-1) of the rapid dissociation phase was comparable to the dissociation rate constant determined in association experiments. A biphasic loss of soluble T1K binding activity was observed with storage at 10 degrees C.