Vasopressin binding sites in toad bladder: studies with the photoaffinity analogue [Phe(p-N3)3]AVP

Toad bladders were exposed to [Phe(p-N3)3]AVP (N3-AVP), an analogue of vasopressin with a photoreactive p-azido group in position three, in the presence and absence of ultraviolet (UV) light. Bladders exposed to the analogue in the presence of UV light showed an increase in membrane permeability to water, which persisted in spite of repeated and prolonged washout of analogue. In contrast, the hydroosmotic response induced by the analogue in the absence of UV light was readily reversed on washout. Aliquots of a broken epithelial cell preparation, derived from bladders that had been exposed to the analogue in the presence of UV light, bound less tritium-labeled vasopressin ([3H]AVP) than control aliquots that had been exposed to the analogue in the absence of UV irradiation or irradiated in the absence of the analogue. Membrane preparations that had not been photolabeled had specific binding sites for [3H]AVP in excess of 1,800 fmol/mg protein without evidence of saturation at a [3H]AVP concentration of 250 nM. Conversely, photolabeled membranes were saturated at a [3H]AVP concentration of 100 nM. The present studies demonstrate that a high proportion of [3H]AVP binding sites can be covalently labeled with N3-AVP and that at least some of these N3-AVP-bound sites are functional in triggering an increase in membrane permeability to water.