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1. A vasopressin (AVP) binding protein was purified from rat liver membranes by an improved method using [125I][d(CH2)5'Sarcosine7]AVP, a selective V1 AVP radioligand and a combination of CHAPS solubilization, gel filtration, lectin affinity and FPLC ion exchange chromatography. 2. The purified protein exhibited a maximum binding activity of 2480 pmol/mg protein with a KD of 4.5 nmol/L, which corresponds to a purification of approximately 26,700-fold. The molecular weight of this protein was 70,000 Da. 3. The binding of [125I][d(CH2)5'Sarcosine7]AVP to the solubilized membranes was dependent on the protein concentration, and was inhibited by the unlabelled peptides [d(CH2)5'Sarcosine7]AVP, AVP, and to a lesser degree by peptides with high V2 receptor affinity, such as 1-desamino-D-AVP and [d(CH2)5'D-Ileu2-Ileu4]AVP. 4. In addition, an AVP anti-idiotypic monoclonal antibody bound to both the partially purified and purified lectin affinity AVP binding protein in a concentration-dependent manner. These results indicate that the purified protein displays similar characteristics to the liver membrane-bound AVP V1 receptor. |
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